Type v crispr/cas effector proteins for cleaving ssdnas and detecting target dnas

ABSTRACT

Provided are compositions and methods for detecting a target DNA (double stranded or single stranded) in a sample. In some embodiments, a subject method includes: (a) contacting the sample with: (i) a type V CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e); (ii) a guide RNA (comprising a region that binds to the type V CRISPR/Cas effector protein, and a guide sequence that hybridizes with the target DNA); and (iii) a detector DNA that is single stranded (i.e., a “single stranded detector DNA”) and does not hybridize with the guide sequence of the guide RNA; and (b) measuring a detectable signal produced by cleavage (by the type V CRISPR/Cas effector protein) of the single stranded detector DNA. Also provided are compositions and methods for cleaving single stranded DNAs (e.g., non-target ssDNAs), e.g., inside of a cell.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/590,106, filed Nov. 22, 2017, and U.S. ProvisionalPatent Application No. 62/626,593, filed Feb. 5, 2018, whichapplications are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. 0950971awarded by the National Science Foundation. The government has certainrights in the invention.

INTRODUCTION

Bacterial adaptive immune systems employ CRISPRs (clustered regularlyinterspaced short palindromic repeats) and CRISPR-associated (Cas)proteins for RNA-guided nucleic acid cleavage. The CRISPR-Cas systemsthereby confer adaptive immunity in bacteria and archaea via RNA-guidednucleic acid interference. To provide anti-viral immunity, processedCRISPR array transcripts (crRNAs) assemble with Cas protein-containingsurveillance complexes that recognize nucleic acids bearing sequencecomplementarity to the virus derived segment of the crRNAs, known as thespacer.

Class 2 CRISPR-Cas systems are streamlined versions in which a singleCas protein (an effector protein, e.g., a type V Cas effector proteinsuch as Cpf1) bound to RNA is responsible for binding to and cleavage ofa targeted sequence. The programmable nature of these minimal systemshas facilitated their use as a versatile technology that continues torevolutionize the field of genome manipulation.

SUMMARY

Class 2 CRISPR-Cas systems (e.g., type V CRISPR/Cas systems such asCas12 family systems) are characterized by effector modules that includea single effector protein. For example, in a type V CRISPR/Cas system,the effector protein—a CRISPR/Cas endonuclease (e.g., a Cas12aprotein)—interacts with (binds to) a corresponding guide RNA (e.g., aCas12a guide RNA) to form a ribonucleoprotein (RNP) complex that istargeted to a particular site in a target nucleic acid via base pairingbetween the guide RNA and a target sequence within the target nucleicacid molecule.

The present disclosure provides compositions and methods that takeadvantage of the discovery that type V CRISPR/Cas proteins (e.g., Cas 12proteins such as Cpf1 (Cas12a) and C2c1 (Cas12b)) can promiscuouslycleave non-targeted single stranded DNA (ssDNA) once activated bydetection of a target DNA. Once a type V CRISPR/Cas effector protein(e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e)is activated by a guide RNA, which occurs when a sample includes atarget DNA to which the guide RNA hybridizes (i.e., the sample includesthe targeted DNA), the protein becomes a nuclease that promiscuouslycleaves ssDNAs (i.e., non-target ssDNAs, i.e., ssDNAs to which the guidesequence of the guide RNA does not hybridize). Thus, when the targetedDNA (double or single stranded) is present in the sample (e.g., in somecases above a threshold amount), the result is cleavage of ssDNAs in thesample, which can be detected using any convenient detection method(e.g., using a labeled single stranded detector DNA).

Provided are compositions and methods for detecting a target DNA (doublestranded or single stranded) in a sample. In some cases, a subjectmethod includes: (a) contacting the sample with: (i) a type V CRISPR/Caseffector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c,Cas12d, Cas12e); (ii) a guide RNA (comprising a region that binds to thetype V CRISPR/Cas effector protein, and a guide sequence that hybridizeswith the target DNA); and (iii) a detector DNA that is single stranded(i.e., a “single stranded detector DNA”) and does not hybridize with theguide sequence of the guide RNA; and (b) measuring a detectable signalproduced by cleavage (by the type V CRISPR/Cas effector protein) of thesingle stranded detector DNA. In some cases, the single strandeddetector DNA includes a fluorescence-emitting dye pair (e.g., afluorescence-emitting dye pair is a fluorescence resonance energytransfer (FRET) pair, a quencher/fluor pair). In some cases, the targetDNA is a viral DNA (e.g., papovavirus, hepadnavirus, herpesvirus,adenovirus, poxvirus, parvovirus, and the like).

Also provided are compositions and methods for cleaving single strandedDNAs (ssDNAs). In some cases, such methods include contacting apopulation of nucleic acids, wherein said population comprises a targetDNA and a plurality of non-target ssDNAs, with: (i) a type V CRISPR/Caseffector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c,Cas12d, Cas12e); and (ii) a guide RNA (comprising a region that binds tothe type V CRISPR/Cas effector protein, and a guide sequence thathybridizes with the target DNA), where the type V CRISPR/Cas effectorprotein cleaves non-target ssDNAs of said plurality. In some cases, thecontacting is inside of a cell such as a eukaryotic cell, a plant cell,a mammalian cell, etc. (e.g., in vitro, ex vivo, in vivo).

Also provided are compositions (e.g., kits) for practicing the subjectmethods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides amino acid sequences of various Type V CRISPR/Caseffector proteins (depicted are Cas12a and Cas12b sequences).

FIG. 2 provides example guide RNA sequences (e.g., crRNA repeatsequences and an example single guide RNA sequence) and example PAMsequences.

FIG. 3 presents data related to non-complementary strand cleavage.

FIG. 4 presents data related to non-specific DNase activity by Cas12a.

FIG. 5 presents data related to non-target strand cleavage.

FIG. 6 presents data related to showing that the RuvC nuclease isresponsible for trans-cleavage of ssDNA.

FIG. 7 presents data related to rapid “shredding” of M13 phage ssDNA.

FIG. 8 presents data related to detection using an FQ-based assay.

FIG. 9 presents data related to mismatches at the PAM-proximal end.

FIG. 10 presents data related to turnover kinetics.

FIG. 11 presents data related to distinguishing viral serotypes using asubject method of detection.

FIG. 12 presents a schematic model for DNA cleavage by CRISPR-Cas12a.

FIG. 13 (panels A-C) presents data showing that Cas12a targetrecognition activates non-specific single stranded DNA cleavage. (panelA) Cartoon of the Cas12a-crRNA complex targeting a dsDNA substrate withcleavage sites depicting the 5′overhang staggered cut. (panel B)Timecourse of purified LbaCas12a targeting the circular, single-strandedM13 DNA phage in vitro reveals a robust shredding pattern. (panel C)Timecourse of purified SpyCas9 targeting M13 ssDNA phage.

FIG. 14 (panels A-C) presents data showing that Cas12a trans-cleavageactivity requires a complementary activator. (panel A) Radiolabeledtarget dsDNA or (panel B) non-specific ssDNA incubated with molar ratiosof LbaCas12a-crRNA as indicated. Each point represents quantified %cleavage after 30-minutes at 37 C, when the reaction was at completion.(panel C) Michaelis-Menten kinetics of LbCas12a trans-cleavage using adsDNA or ssDNA activator.

FIG. 15 (panels A-C) presents data showing that specificity oftrans-cleavage activation involves PAM recognition and DNA unwinding.(panel A) Trans-cleavage products on a denaturing PAGE gel with theindicated activators. (panel B) Observed trans-cleavage rates using assDNA or dsDNA activator with indicated mismatches. (panel C) LbaCas12acan distinguish two closely related dsDNA HPV sequences.

FIG. 16 (panels A-C) presents data showing that non-specific ssDNAcleavage activity is conserved across Type V CRISPR systems. (panel A)Phylogenetic tree highlighting indicated type V effector proteins.(panel B) Cleavage gels depicting activator-dependent trans-cleavageacross type V effector proteins, but not the type II effector SpyCas9.(panel C) Model for PAM-dependent and PAM-independent activation of cisand trans-cleavage.

FIG. 17 presents data showing that target strand recognition is apre-requisite for single-stranded DNA cleavage.

FIG. 18 (panels A-C) presents data showing that the RuvC nuclease isresponsible for non-specific DNase activity.

FIG. 19 presents data showing that the circular, single-stranded M13 DNAphage is degraded in trans by a pre-activated LbaCas12a complex.

FIG. 20 (panels A-B) presents data showing that LbaCas12a is activatedby a dsDNA plasmid for trans-cleavage.

FIG. 21 presents data showing that LbaCas12a trans-cleavage degradescomplementary and non-specific ssDNA, but not ssRNA.

FIG. 22 presents data showing that Michaelis-Menten kinetics revealsrobust trans-cleavage activity with a ssDNA and dsDNA activator.

FIG. 23 presents data showing that the PAM sequence and PAM-proximalmismatches in a dsDNA activator provide specificity fortrans-activation.

FIG. 24 presents data showing an HPV detection assay timecourse detectedusing a subject method of detection (e.g., labeled detector ssDNA).

FIG. 25 (panels A-C) presents data showing that Cas12a targetrecognition activates non-specific single-stranded DNA cleavage.

FIG. 26 (panels A-D) presents data related to kinetics of Cas12a ssDNAtrans-cleavage.

FIG. 27 (panels A-C) presents data showing specificity and conservationof trans-cleavage activation.

FIG. 28 (panels A-D) presents data showing rapid identification of HPVtypes 16 and 18 in human samples by DETECTR.

FIG. 29 presents a schematic model for PAM-dependent and PAM-independentactivation of cis and trans-cleavage by Cas12a.

FIG. 30 presents data showing purification of Cas12 and Cas9 proteins.

FIG. 31 (panels A-B) presents data showing that LbCas12a is aDNA-activated general DNase.

FIG. 32 (panels A-B) presents data showing that target strandrecognition is a pre-requisite for single-stranded DNA cleavage

FIG. 33 (panels A-C) presents data showing that the RuvC nuclease domainis responsible for activator-dependent, non-specific DNase activity.

FIG. 34 (panels A-C) presents data showing that LbCas12a trans-cleavagedegrades complementary and non-specific ssDNA, but not ssRNA.

FIG. 35 (panels A-B) presents data showing that target strand cleavageby Cas12a is not required for triggering non-specific ssDNase activity.

FIG. 36 (panels A-E) presents data showing Michaelis-Menten analysisthat reveals robust trans-cleavage activity with a ssDNA and dsDNAactivator.

FIG. 37 presents data showing that PAM sequence and PAM-proximalmismatches in a dsDNA activator provide specificity fortrans-activation.

FIG. 38 presents data showing that activator-dependent, non-specificssDNA cleavage activity is conserved across type V CRISPR interferenceproteins.

FIG. 39 (panels A-E) presents data showing that Cas12a can distinguishtwo closely related HPV sequences.

FIG. 40 (panels A-B) presents data showing that isothermal amplificationcoupled with Cas12a detection yields DETECTR, which can achieveattomolar sensitivity

FIG. 41 (panels A-D) presents data showing identification of HPV types16 and 18 in human cell lines and patient samples by DETECTR

FIG. 42 (panels A-B) presents data showing PCR and hybrid capturevalidation and genotyping of HPV in human clinical samples.

FIG. 43 presents data showing identification of target nucleic acid byDETECTR using Cas12d and Cas12e proteins.

FIG. 44 presents data showing identification of a single nucleotidepolymorphism (SNP) within the HERC2 gene responsible for brown or blueeyes using DETECTR.

FIG. 45 presents data showing identification of the X or Y chromosomesthrough detection of the XIST (within X chromosome) or SRY (within Ychromosome) genes from human saliva (using the DETECTR assay).

FIG. 46 presents a schematic illustrating DETECTR as a platform forrapid, point-of-care diagnostics.

DEFINITIONS

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides.

Thus, terms “polynucleotide” and “nucleic acid” encompasssingle-stranded DNA; double-stranded DNA; multi-stranded DNA;single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomicDNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

The term “oligonucleotide” refers to a polynucleotide of between 4 and100 nucleotides of single- or double-stranded nucleic acid (e.g., DNA,RNA, or a modified nucleic acid). However, for the purposes of thisdisclosure, there is no upper limit to the length of an oligonucleotide.Oligonucleotides are also known as “oligomers” or “oligos” and can beisolated from genes, transcribed (in vitro and/or in vivo), orchemically synthesized. The terms “polynucleotide” and “nucleic acid”should be understood to include, as applicable to the embodiments beingdescribed, single-stranded (such as sense or antisense) anddouble-stranded polynucleotides.

By “hybridizable” or “complementary” or “substantially complementary” itis meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence ofnucleotides that enables it to non-covalently bind, i.e. formWatson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,”to another nucleic acid in a sequence-specific, antiparallel, manner(i.e., a nucleic acid specifically binds to a complementary nucleicacid) under the appropriate in vitro and/or in vivo conditions oftemperature and solution ionic strength. Standard Watson-Crickbase-pairing includes: adenine/adenosine) (A) pairing withthymidine/thymidine (T), A pairing with uracil/uridine (U), andguanine/guanosine) (G) pairing with cytosine/cytidine (C). In addition,for hybridization between two RNA molecules (e.g., dsRNA), and forhybridization of a DNA molecule with an RNA molecule (e.g., when a DNAtarget nucleic acid base pairs with a guide RNA, etc.): G can also basepair with U. For example, G/U base-pairing is partially responsible forthe degeneracy (i.e., redundancy) of the genetic code in the context oftRNA anti-codon base-pairing with codons in mRNA. Thus, in the contextof this disclosure, a G (e.g., of a protein-binding segment (e.g., dsRNAduplex) of a guide RNA molecule; of a target nucleic acid (e.g., targetDNA) base pairing with a guide RNA) is considered complementary to botha U and to C. For example, when a G/U base-pair can be made at a givennucleotide position of a protein-binding segment (e.g., dsRNA duplex) ofa guide RNA molecule, the position is not considered to benon-complementary, but is instead considered to be complementary.

Hybridization requires that the two nucleic acids contain complementarysequences, although mismatches between bases are possible. Theconditions appropriate for hybridization between two nucleic acidsdepend on the length of the nucleic acids and the degree ofcomplementarity, variables well known in the art. The greater the degreeof complementarity between two nucleotide sequences, the greater thevalue of the melting temperature (Tm) for hybrids of nucleic acidshaving those sequences. Typically, the length for a hybridizable nucleicacid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

It is understood that the sequence of a polynucleotide need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. Moreover, a polynucleotide may hybridize over one or moresegments such that intervening or adjacent segments are not involved inthe hybridization event (e.g., a loop structure or hairpin structure, a‘bulge’, and the like). A polynucleotide can comprise 60% or more, 65%or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% ormore, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%sequence complementarity to a target region within the target nucleicacid sequence to which it will hybridize. For example, an antisensenucleic acid in which 18 of 20 nucleotides of the antisense compound arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. The remainingnoncomplementary nucleotides may be clustered or interspersed withcomplementary nucleotides and need not be contiguous to each other or tocomplementary nucleotides. Percent complementarity between particularstretches of nucleic acid sequences within nucleic acids can bedetermined using any convenient method. Example methods include BLASTprograms (basic local alignment search tools) and PowerBLAST programs(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656) or by using the Gap program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, Madison Wis.), e.g., using default settings,which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981,2, 482-489).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

“Binding” as used herein (e.g. with reference to an RNA-binding domainof a polypeptide, binding to a target nucleic acid, and the like) refersto a non-covalent interaction between macromolecules (e.g., between aprotein and a nucleic acid; between a guide RNA and a target nucleicacid; and the like). While in a state of non-covalent interaction, themacromolecules are said to be “associated” or “interacting” or “binding”(e.g., when a molecule X is said to interact with a molecule Y, it ismeant the molecule X binds to molecule Y in a non-covalent manner). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), but some portionsof a binding interaction may be sequence-specific. Binding interactionsare generally characterized by a dissociation constant (K_(d)) of lessthan 10⁻⁶ M, less than 10⁻⁷ M, less than 10⁻⁸M, less than 10⁻⁹ M, lessthan 10⁻¹⁰ M, less than 10⁻¹¹ M, less than 10⁻¹⁻² M, less than 10⁻¹³ M,less than 10⁻¹⁴ M, or less than 10⁻¹⁵ M. “Affinity” refers to thestrength of binding, increased binding affinity being correlated with alower K_(d).

By “binding domain” it is meant a protein domain that is able to bindnon-covalently to another molecule. A binding domain can bind to, forexample, an RNA molecule (an RNA-binding domain) and/or a proteinmolecule (a protein-binding domain). In the case of a protein having aprotein-binding domain, it can in some cases bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or more regionsof a different protein or proteins.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains. For example, a group of amino acids having aliphatic sidechains consists of glycine, alanine, valine, leucine, and isoleucine; agroup of amino acids having aliphatic-hydroxyl side chains consists ofserine and threonine; a group of amino acids having amide containingside chains consisting of asparagine and glutamine; a group of aminoacids having aromatic side chains consists of phenylalanine, tyrosine,and tryptophan; a group of amino acids having basic side chains consistsof lysine, arginine, and histidine; a group of amino acids having acidicside chains consists of glutamate and aspartate; and a group of aminoacids having sulfur containing side chains consists of cysteine andmethionine. Exemplary conservative amino acid substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine-glycine, and asparagine-glutamine.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same, and inthe same relative position, when comparing the two sequences. Sequenceidentity can be determined in a number of different ways. To determinesequence identity, sequences can be aligned using various methods andcomputer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.),available over the world wide web at sites includingncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/,ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/,http://www.sbg.bio.ic.ac.uk/˜phyre2/. See, e.g., Altschul et al. (1990),J. Mol. Bioi. 215:403-10.

The terms “DNA regulatory sequences,” “control elements,” and“regulatory elements,” used interchangeably herein, refer totranscriptional and translational control sequences, such as promoters,enhancers, polyadenylation signals, terminators, protein degradationsignals, and the like, that provide for and/or regulate transcription ofa non-coding sequence (e.g., guide RNA) or a coding sequence (e.g.,protein coding) and/or regulate translation of an encoded polypeptide.

As used herein, a “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of a downstream(3′ direction) coding or non-coding sequence. Eukaryotic promoters willoften, but not always, contain “TATA” boxes and “CAT” boxes. Variouspromoters, including inducible promoters, may be used to drive thevarious nucleic acids (e.g., vectors) of the present disclosure.

The term “naturally-occurring” or “unmodified” or “wild type” as usedherein as applied to a nucleic acid, a polypeptide, a cell, or anorganism, refers to a nucleic acid, polypeptide, cell, or organism thatis found in nature.

“Recombinant,” as used herein, means that a particular nucleic acid (DNAor RNA) is the product of various combinations of cloning, restriction,polymerase chain reaction (PCR) and/or ligation steps resulting in aconstruct having a structural coding or non-coding sequencedistinguishable from endogenous nucleic acids found in natural systems.DNA sequences encoding polypeptides can be assembled from cDNA fragmentsor from a series of synthetic oligonucleotides, to provide a syntheticnucleic acid which is capable of being expressed from a recombinanttranscriptional unit contained in a cell or in a cell-free transcriptionand translation system. Genomic DNA comprising the relevant sequencescan also be used in the formation of a recombinant gene ortranscriptional unit. Sequences of non-translated DNA may be present 5′or 3′ from the open reading frame, where such sequences do not interferewith manipulation or expression of the coding regions, and may indeedact to modulate production of a desired product by various mechanisms(see “DNA regulatory sequences”, below). Alternatively, DNA sequencesencoding RNA (e.g., guide RNA) that is not translated may also beconsidered recombinant. Thus, e.g., the term “recombinant” nucleic acidrefers to one which is not naturally occurring, e.g., is made by theartificial combination of two otherwise separated segments of sequencethrough human intervention. This artificial combination is oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques. Such is usually done to replace a codon with acodon encoding the same amino acid, a conservative amino acid, or anon-conservative amino acid. Alternatively, it is performed to jointogether nucleic acid segments of desired functions to generate adesired combination of functions. This artificial combination is oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques. When a recombinant polynucleotide encodes apolypeptide, the sequence of the encoded polypeptide can be naturallyoccurring (“wild type”) or can be a variant (e.g., a mutant) of thenaturally occurring sequence. Thus, the term “recombinant” polypeptidedoes not necessarily refer to a polypeptide whose sequence does notnaturally occur. Instead, a “recombinant” polypeptide is encoded by arecombinant DNA sequence, but the sequence of the polypeptide can benaturally occurring (“wild type”) or non-naturally occurring (e.g., avariant, a mutant, etc.). Thus, a “recombinant” polypeptide is theresult of human intervention, but may be a naturally occurring aminoacid sequence.

A “vector” or “expression vector” is a replicon, such as plasmid, phage,virus, or cosmid, to which another DNA segment, i.e. an “insert”, may beattached so as to bring about the replication of the attached segment ina cell.

An “expression cassette” comprises a DNA coding sequence operably linkedto a promoter. “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner For instance, a promoter is operablylinked to a coding sequence if the promoter affects its transcription orexpression.

The terms “recombinant expression vector,” or “DNA construct” are usedinterchangeably herein to refer to a DNA molecule comprising a vectorand one insert. Recombinant expression vectors are usually generated forthe purpose of expressing and/or propagating the insert(s), or for theconstruction of other recombinant nucleotide sequences. The insert(s)may or may not be operably linked to a promoter sequence and may or maynot be operably linked to DNA regulatory sequences.

Any given component, or combination of components can be unlabeled, orcan be detectably labeled with a label moiety. In some cases, when twoor more components are labeled, they can be labeled with label moietiesthat are distinguishable from one another.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “atype V CRISPR/Cas effector protein” includes a plurality of such type VCRISPR/Cas effector proteins and reference to “the guide RNA” includesreference to one or more guide RNAs and equivalents thereof known tothose skilled in the art, and so forth. It is further noted that theclaims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for use of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

As noted above, the inventors have discovered that that type VCRISPR/Cas proteins, e.g., Cas12 proteins such as Cpf1 (Cas12a) and C2c1(Cas12b) can promiscuously cleave non-targeted single stranded DNA(ssDNA) once activated by detection of a target DNA (double or singlestranded). Once a type V CRISPR/Cas effector protein (e.g., a Cas12protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) is activated bya guide RNA, which occurs when the guide RNA hybridizes to a targetsequence of a target DNA (i.e., the sample includes the targeted DNA),the protein becomes a nuclease that promiscuously cleaves ssDNAs (i.e.,the nuclease cleaves non-target ssDNAs, i.e., ssDNAs to which the guidesequence of the guide RNA does not hybridize). Thus, when the target DNAis present in the sample (e.g., in some cases above a threshold amount),the result is cleavage of ssDNAs in the sample, which can be detectedusing any convenient detection method (e.g., using a labeled singlestranded detector DNA).

Provided are compositions and methods for detecting a target DNA (doublestranded or single stranded) in a sample. In some cases, a detector DNAis used that is single stranded (ssDNA) and does not hybridize with theguide sequence of the guide RNA (i.e., the detector ssDNA is anon-target ssDNA). Such methods can include (a) contacting the samplewith: (i) a type V CRISPR/Cas effector protein (e.g., a Cas12 protein);(ii) a guide RNA comprising: a region that binds to the type VCRISPR/Cas effector protein, and a guide sequence that hybridizes withthe target DNA; and (iii) a detector DNA that is single stranded anddoes not hybridize with the guide sequence of the guide RNA; and (b)measuring a detectable signal produced by cleavage of the singlestranded detector DNA by the type V CRISPR/Cas effector protein, therebydetecting the target DNA. As noted above, once a subject Type VCRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a,Cas12b, Cas12c, Cas12d, Cas12e) is activated by a guide RNA, whichoccurs when the sample includes a target DNA to which the guide RNAhybridizes (i.e., the sample includes the targeted target DNA), the TypeV CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a,Cas12b, Cas12c, Cas12d, Cas12e) is activated and functions as anendoribonuclease that non-specifically cleaves ssDNAs (includingnon-target ssDNAs) present in the sample. Thus, when the targeted targetDNA is present in the sample (e.g., in some cases above a thresholdamount), the result is cleavage of ssDNA (including non-target ssDNA) inthe sample, which can be detected using any convenient detection method(e.g., using a labeled detector ssDNA).

Also provided are compositions and methods for cleaving single strandedDNAs (ssDNAs) (e.g., non-target ssDNAs). Such methods can includecontacting a population of nucleic acids, wherein said populationcomprises a target DNA and a plurality of non-target ssDNAs, with: (i) atype V CRISPR/Cas effector protein; and (ii) a guide RNA comprising: aregion that binds to the type V CRISPR/Cas effector protein, and a guidesequence that hybridizes with the target DNA, wherein the type VCRISPR/Cas effector protein cleaves non-target ssDNAs of said plurality.Such a method can be used, e.g., to cleave foreign ssDNAs (e.g., viralDNAs) in a cell.

The contacting step of a subject method can be carried out in acomposition comprising divalent metal ions. The contacting step can becarried out in an acellular environment, e.g., outside of a cell. Thecontacting step can be carried out inside a cell. The contacting stepcan be carried out in a cell in vitro. The contacting step can becarried out in a cell ex vivo. The contacting step can be carried out ina cell in vivo.

The guide RNA can be provided as RNA or as a nucleic acid encoding theguide RNA (e.g., a DNA such as a recombinant expression vector). TheType V CRISPR/Cas effector protein (e.g., a Cas12 protein such asCas12a, Cas12b, Cas12c, Cas12d, Cas12e) can be provided as a protein oras a nucleic acid encoding the protein (e.g., an mRNA, a DNA such as arecombinant expression vector). In some cases, two or more (e.g., 3 ormore, 4 or more, 5 or more, or 6 or more) guide RNAs can be provided by(e.g., using a precursor guide RNA array, which can be cleaved by theType V CRISPR/Cas effector protein into individual (“mature”) guideRNAs).

In some cases (e.g., when contacting with a guide RNA and a Type VCRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a,Cas12b, Cas12c, Cas12d, Cas12e)), the sample is contacted for 2 hours orless (e.g., 1.5 hours or less, 1 hour or less, 40 minutes or less, 30minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes orless, or 1 minute or less) prior to the measuring step. For example, insome cases the sample is contacted for 40 minutes or less prior to themeasuring step. In some cases the sample is contacted for 20 minutes orless prior to the measuring step. In some cases the sample is contactedfor 10 minutes or less prior to the measuring step. In some cases thesample is contacted for 5 minutes or less prior to the measuring step.In some cases the sample is contacted for 1 minute or less prior to themeasuring step. In some cases the sample is contacted for from 50seconds to 60 seconds prior to the measuring step. In some cases thesample is contacted for from 40 seconds to 50 seconds prior to themeasuring step. In some cases the sample is contacted for from 30seconds to 40 seconds prior to the measuring step. In some cases thesample is contacted for from 20 seconds to 30 seconds prior to themeasuring step. In some cases the sample is contacted for from 10seconds to 20 seconds prior to the measuring step.

A method of the present disclosure for detecting a target DNA(single-stranded or double-stranded) in a sample can detect a target DNAwith a high degree of sensitivity. In some cases, a method of thepresent disclosure can be used to detect a target DNA present in asample comprising a plurality of DNAs (including the target DNA and aplurality of non-target DNAs), where the target DNA is present at one ormore copies per 10⁷ non-target DNAs (e.g., one or more copies per 10⁶non-target DNAs, one or more copies per 10⁵ non-target DNAs, one or morecopies per 10⁴ non-target DNAs, one or more copies per 10³ non-targetDNAs, one or more copies per 10² non-target DNAs, one or more copies per50 non-target DNAs, one or more copies per 20 non-target DNAs, one ormore copies per 10 non-target DNAs, or one or more copies per 5non-target DNAs). In some cases, a method of the present disclosure canbe used to detect a target DNA present in a sample comprising aplurality of DNAs (including the target DNA and a plurality ofnon-target DNAs), where the target DNA is present at one or more copiesper 10¹⁸ non-target DNAs (e.g., one or more copies per 10¹⁵ non-targetDNAs, one or more copies per 10¹² non-target DNAs, one or more copiesper 10⁹ non-target DNAs, one or more copies per 10⁶ non-target DNAs, oneor more copies per 10⁵ non-target DNAs, one or more copies per 10⁴non-target DNAs, one or more copies per 10³ non-target DNAs, one or morecopies per 10² non-target DNAs, one or more copies per 50 non-targetDNAs, one or more copies per 20 non-target DNAs, one or more copies per10 non-target DNAs, or one or more copies per 5 non-target DNAs).

In some cases, a method of the present disclosure can detect a targetDNA present in a sample, where the target DNA is present at from onecopy per 10⁷ non-target DNAs to one copy per 10 non-target DNAs (e.g.,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁶ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10 non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10 non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10³ non-target DNAs,or from 1 copy per 10⁵ non-target DNAs to 1 copy per 10⁴ non-targetDNAs).

In some cases, a method of the present disclosure can detect a targetDNA present in a sample, where the target DNA is present at from onecopy per 10¹⁸ non-target DNAs to one copy per 10 non-target DNAs (e.g.,from 1 copy per 10¹⁸ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10¹⁵ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10¹² non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁹ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁶ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10 non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10 non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10³ non-target DNAs,or from 1 copy per 10⁵ non-target DNAs to 1 copy per 10⁴ non-targetDNAs).

In some cases, a method of the present disclosure can detect a targetDNA present in a sample, where the target DNA is present at from onecopy per 10⁷ non-target DNAs to one copy per 100 non-target DNAs (e.g.,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁶ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 100 non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 100 non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10³ non-target DNAs,or from 1 copy per 10⁵ non-target DNAs to 1 copy per 10⁴ non-targetDNAs).

In some cases, the threshold of detection, for a subject method ofdetecting a target DNA in a sample, is 10 nM or less. The term“threshold of detection” is used herein to describe the minimal amountof target DNA that must be present in a sample in order for detection tooccur. Thus, as an illustrative example, when a threshold of detectionis 10 nM, then a signal can be detected when a target DNA is present inthe sample at a concentration of 10 nM or more. In some cases, a methodof the present disclosure has a threshold of detection of 5 nM or less.In some cases, a method of the present disclosure has a threshold ofdetection of 1 nM or less. In some cases, a method of the presentdisclosure has a threshold of detection of 0.5 nM or less. In somecases, a method of the present disclosure has a threshold of detectionof 0.1 nM or less. In some cases, a method of the present disclosure hasa threshold of detection of 0.05 nM or less. In some cases, a method ofthe present disclosure has a threshold of detection of 0.01 nM or less.In some cases, a method of the present disclosure has a threshold ofdetection of 0.005 nM or less. In some cases, a method of the presentdisclosure has a threshold of detection of 0.001 nM or less. In somecases, a method of the present disclosure has a threshold of detectionof 0.0005 nM or less. In some cases, a method of the present disclosurehas a threshold of detection of 0.0001 nM or less. In some cases, amethod of the present disclosure has a threshold of detection of 0.00005nM or less. In some cases, a method of the present disclosure has athreshold of detection of 0.00001 nM or less. In some cases, a method ofthe present disclosure has a threshold of detection of 10 pM or less. Insome cases, a method of the present disclosure has a threshold ofdetection of 1 pM or less. In some cases, a method of the presentdisclosure has a threshold of detection of 500 fM or less. In somecases, a method of the present disclosure has a threshold of detectionof 250 fM or less. In some cases, a method of the present disclosure hasa threshold of detection of 100 fM or less. In some cases, a method ofthe present disclosure has a threshold of detection of 50 fM or less. Insome cases, a method of the present disclosure has a threshold ofdetection of 500 aM (attomolar) or less. In some cases, a method of thepresent disclosure has a threshold of detection of 250 aM or less. Insome cases, a method of the present disclosure has a threshold ofdetection of 100 aM or less. In some cases, a method of the presentdisclosure has a threshold of detection of 50 aM or less. In some cases,a method of the present disclosure has a threshold of detection of 10 aMor less. In some cases, a method of the present disclosure has athreshold of detection of 1 aM or less.

In some cases, the threshold of detection (for detecting the target DNAin a subject method), is in a range of from 500 fM to 1 nM (e.g., from500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500fM to 10 pM, from 500 fM to 1 pM, from 800 fM to 1 nM, from 800 fM to500 pM, from 800 fM to 200 pM, from 800 fM to 100 pM, from 800 fM to 10pM, from 800 fM to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1pM to 200 pM, from 1 pM to 100 pM, or from 1 pM to 10 pM) (where theconcentration refers to the threshold concentration of target DNA atwhich the target DNA can be detected). In some cases, a method of thepresent disclosure has a threshold of detection in a range of from 800fM to 100 pM. In some cases, a method of the present disclosure has athreshold of detection in a range of from 1 pM to 10 pM. In some cases,a method of the present disclosure has a threshold of detection in arange of from 10 fM to 500 fM, e.g., from 10 fM to 50 fM, from 50 fM to100 fM, from 100 fM to 250 fM, or from 250 fM to 500 fM.

In some cases, the minimum concentration at which a target DNA can bedetected in a sample is in a range of from 500 fM to 1 nM (e.g., from500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500fM to 10 pM, from 500 fM to 1 pM, from 800 fM to 1 nM, from 800 fM to500 pM, from 800 fM to 200 pM, from 800 fM to 100 pM, from 800 fM to 10pM, from 800 fM to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1pM to 200 pM, from 1 pM to 100 pM, or from 1 pM to 10 pM). In somecases, the minimum concentration at which a target DNA can be detectedin a sample is in a range of from 800 fM to 100 pM. In some cases, theminimum concentration at which a target DNA can be detected in a sampleis in a range of from 1 pM to 10 pM.

In some cases, the threshold of detection (for detecting the target DNAin a subject method), is in a range of from 1 aM to 1 nM (e.g., from 1aM to 500 pM, from 1 aM to 200 pM, from 1 aM to 100 pM, from 1 aM to 10pM, from 1 aM to 1 pM, from 100 aM to 1 nM, from 100 aM to 500 pM, from100 aM to 200 pM, from 100 aM to 100 pM, from 100 aM to 10 pM, from 100aM to 1 pM, from 250 aM to 1 nM, from 250 aM to 500 pM, from 250 aM to200 pM, from 250 aM to 100 pM, from 250 aM to 10 pM, from 250 aM to 1pM, from 500 aM to 1 nM, from 500 aM to 500 pM, from 500 aM to 200 pM,from 500 aM to 100 pM, from 500 aM to 10 pM, from 500 aM to 1 pM, from750 aM to 1 nM, from 750 aM to 500 pM, from 750 aM to 200 pM, from 750aM to 100 pM, from 750 aM to 10 pM, from 750 aM to 1 pM, from 1 fM to 1nM, from 1 fM to 500 pM, from 1 fM to 200 pM, from 1 fM to 100 pM, from1 fM to 10 pM, from 1 fM to 1 pM, from 500 fM to 500 pM, from 500 fM to200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500 fM to 1pM, from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM,from 800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1pM to 1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100pM, or from 1 pM to 10 pM) (where the concentration refers to thethreshold concentration of target DNA at which the target DNA can bedetected). In some cases, a method of the present disclosure has athreshold of detection in a range of from 1 aM to 800 aM. In some cases,a method of the present disclosure has a threshold of detection in arange of from 50 aM to 1 pM. In some cases, a method of the presentdisclosure has a threshold of detection in a range of from 50 aM to 500fM.

In some cases, the minimum concentration at which a target DNA can bedetected in a sample is in a range of from 1 aM to 1 nM (e.g., from 1 aMto 500 pM, from 1 aM to 200 pM, from 1 aM to 100 pM, from 1 aM to 10 pM,from 1 aM to 1 pM, from 100 aM to 1 nM, from 100 aM to 500 pM, from 100aM to 200 pM, from 100 aM to 100 pM, from 100 aM to 10 pM, from 100 aMto 1 pM, from 250 aM to 1 nM, from 250 aM to 500 pM, from 250 aM to 200pM, from 250 aM to 100 pM, from 250 aM to 10 pM, from 250 aM to 1 pM,from 500 aM to 1 nM, from 500 aM to 500 pM, from 500 aM to 200 pM, from500 aM to 100 pM, from 500 aM to 10 pM, from 500 aM to 1 pM, from 750 aMto 1 nM, from 750 aM to 500 pM, from 750 aM to 200 pM, from 750 aM to100 pM, from 750 aM to 10 pM, from 750 aM to 1 pM, from 1 fM to 1 nM,from 1 fM to 500 pM, from 1 fM to 200 pM, from 1 fM to 100 pM, from 1 fMto 10 pM, from 1 fM to 1 pM, from 500 fM to 500 pM, from 500 fM to 200pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500 fM to 1 pM,from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM, from800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1 pMto 1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100 pM,or from 1 pM to 10 pM). In some cases, the minimum concentration atwhich a target DNA can be detected in a sample is in a range of from 1aM to 500 pM. In some cases, the minimum concentration at which a targetDNA can be detected in a sample is in a range of from 100 aM to 500 pM.

In some cases, a subject composition or method exhibits an attomolar(aM) sensitivity of detection. In some cases, a subject composition ormethod exhibits a femtomolar (fM) sensitivity of detection. In somecases, a subject composition or method exhibits a picomolar (pM)sensitivity of detection. In some cases, a subject composition or methodexhibits a nanomolar (nM) sensitivity of detection.

Target DNA

A target DNA can be single stranded (ssDNA) or double stranded (dsDNA).When the target DNA is single stranded, there is no preference orrequirement for a PAM sequence in the target DNA. However, when thetarget DNA is dsDNA, a PAM is usually present adjacent to the targetsequence of the target DNA (e.g., see discussion of the PAM elsewhereherein). The source of the target DNA can be the same as the source ofthe sample, e.g., as described below.

The source of the target DNA can be any source. In some cases the targetDNA is a viral

DNA (e.g., a genomic DNA of a DNA virus). As such, subject method can befor detecting the presence of a viral DNA amongst a population ofnucleic acids (e.g., in a sample). A subject method can also be used forthe cleavage of non-target ssDNAs in the present of a target DNA.example, if a method takes place in a cell, a subject method can be usedto promiscuously cleave non-target ssDNAs in the cell (ssDNAs that donot hybridize with the guide sequence of the guide RNA) when aparticular target DNA is present in the cell (e.g., when the cell isinfected with a virus and viral target DNA is detected).

Examples of possible target DNAs include, but are not limited to, viralDNAs such as: a papovavirus (e.g., human papillomavirus (HPV),polyomavirus); a hepadnavirus (e.g., Hepatitis B Virus (HBV)); aherpesvirus (e.g., herpes simplex virus (HSV), varicella zoster virus(VZV), epstein-barr virus (EBV), cytomegalovirus (CMV), herpeslymphotropic virus, Pityriasis Rosea, kaposi's sarcoma-associatedherpesvirus); an adenovirus (e.g., atadenovirus, aviadenovirus,ichtadenovirus, mastadenovirus, siadenovirus); a poxvirus (e.g.,smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus,pseudocowpox, bovine papular stomatitis virus; tanapox virus, yabamonkey tumor virus; molluscum contagiosum virus (MCV)); a parvovirus(e.g., adeno-associated virus (AAV), Parvovirus B19, human bocavirus,bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; Phycodnaviridae;and the like. In some cases, the target DNA is parasite DNA. In somecases, the target DNA is bacterial DNA, e.g., DNA of a pathogenicbacterium.

Samples

A subject sample includes nucleic acid (e.g., a plurality of nucleicacids). The term “plurality” is used herein to mean two or more. Thus,in some cases a sample includes two or more (e.g., 3 or more, 5 or more,10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 ormore, or 5,000 or more) nucleic acids (e.g., DNAs). A subject method canbe used as a very sensitive way to detect a target DNA present in asample (e.g., in a complex mixture of nucleic acids such as DNAs). Insome cases the sample includes 5 or more DNAs (e.g., 10 or more, 20 ormore, 50 or more, 100 or more, 500 or more, 1,000 or more, or 5,000 ormore DNAs) that differ from one another in sequence. In some cases, thesample includes 10 or more, 20 or more, 50 or more, 100 or more, 500 ormore, 10³ or more, 5×10³ or more, 10⁴ or more, 5×10⁴ or more, 10⁵ ormore, 5×10⁵ or more, 10⁶ or more 5×10⁶ or more, or 10⁷ or more, DNAs. Insome cases, the sample comprises from 10 to 20, from 20 to 50, from 50to 100, from 100 to 500, from 500 to 10³, from 10³ to 5×10³, from 5×10³to 10⁴, from 10⁴ to 5×10⁴, from 5×10⁴ to 10⁵, from 10⁵ to 5×10⁵, from5×10⁵ to 10⁶, from 10⁶ to 5×10⁶, or from 5×10⁶ to 10⁷, or more than 10⁷,DNAs. In some cases, the sample comprises from 5 to 10⁷ DNAs (e.g., thatdiffer from one another in sequence)(e.g., from 5 to 10⁶, from 5 to 10⁵,from 5 to 50,000, from 5 to 30,000, from 10 to 10⁶, from 10 to 10⁵, from10 to 50,000, from 10 to 30,000, from 20 to 10⁶, from 20 to 10⁵, from 20to 50,000, or from 20 to 30,000 DNAs). In some cases the sample includes20 or more DNAs that differ from one another in sequence. In some cases,the sample includes DNAs from a cell lysate (e.g., a eukaryotic celllysate, a mammalian cell lysate, a human cell lysate, a prokaryotic celllysate, a plant cell lysate, and the like). For example, in some casesthe sample includes DNA from a cell such as a eukaryotic cell, e.g., amammalian cell such as a human cell.

The term “sample” is used herein to mean any sample that includes DNA(e.g., in order to determine whether a target DNA is present among apopulation of DNAs). The sample can be derived from any source, e.g.,the sample can be a synthetic combination of purified DNAs; the samplecan be a cell lysate, an DNA-enriched cell lysate, or DNAs isolatedand/or purified from a cell lysate. The sample can be from a patient(e.g., for the purpose of diagnosis). The sample can be frompermeabilized cells. The sample can be from crosslinked cells. Thesample can be in tissue sections. The sample can be from tissuesprepared by crosslinking followed by delipidation and adjustment to makea uniform refractive index. Examples of tissue preparation bycrosslinking followed by delipidation and adjustment to make a uniformrefractive index have been described in, for example, Shah et al.,Development (2016) 143, 2862-2867 doi:10.1242/dev.138560.

A “sample” can include a target DNA and a plurality of non-target DNAs.In some cases, the target DNA is present in the sample at one copy per10 non-target DNAs, one copy per 20 non-target DNAs, one copy per 25non-target DNAs, one copy per 50 non-target DNAs, one copy per 100non-target DNAs, one copy per 500 non-target DNAs, one copy per 10³non-target DNAs, one copy per 5×10³ non-target DNAs, one copy per 10⁴non-target DNAs, one copy per 5×10⁴ non-target DNAs, one copy per 10⁵non-target DNAs, one copy per 5×10⁵ non-target DNAs, one copy per 10⁶non-target DNAs, or less than one copy per 10⁶ non-target DNAs. In somecases, the target DNA is present in the sample at from one copy per 10non-target DNAs to 1 copy per 20 non-target DNAs, from 1 copy per 20non-target DNAs to 1 copy per 50 non-target DNAs, from 1 copy per 50non-target DNAs to 1 copy per 100 non-target DNAs, from 1 copy per 100non-target DNAs to 1 copy per 500 non-target DNAs, from 1 copy per 500non-target DNAs to 1 copy per 10³ non-target DNAs, from 1 copy per 10³non-target DNAs to 1 copy per 5×10³ non-target DNAs, from 1 copy per5×10³ non-target DNAs to 1 copy per 10⁴ non-target DNAs, from 1 copy per10⁴ non-target DNAs to 1 copy per 10⁵ non-target DNAs, from 1 copy per10⁵ non-target DNAs to 1 copy per 10⁶ non-target DNAs, or from 1 copyper 10⁶ non-target DNAs to 1 copy per 10⁷ non-target DNAs.

Suitable samples include but are not limited to saliva, blood, serum,plasma, urine, aspirate, and biopsy samples. Thus, the term “sample”with respect to a patient encompasses blood and other liquid samples ofbiological origin, solid tissue samples such as a biopsy specimen ortissue cultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents; washed; orenrichment for certain cell populations, such as cancer cells. Thedefinition also includes sample that have been enriched for particulartypes of molecules, e.g., DNAs. The term “sample” encompasses biologicalsamples such as a clinical sample such as blood, plasma, serum,aspirate, cerebral spinal fluid (CSF), and also includes tissue obtainedby surgical resection, tissue obtained by biopsy, cells in culture, cellsupernatants, cell lysates, tissue samples, organs, bone marrow, and thelike. A “biological sample” includes biological fluids derived therefrom(e.g., cancerous cell, infected cell, etc.), e.g., a sample comprisingDNAs that is obtained from such cells (e.g., a cell lysate or other cellextract comprising DNAs).

A sample can comprise, or can be obtained from, any of a variety ofcells, tissues, organs, or acellular fluids. Suitable sample sourcesinclude eukaryotic cells, bacterial cells, and archaeal cells. Suitablesample sources include single-celled organisms and multi-cellularorganisms. Suitable sample sources include single-cell eukaryoticorganisms; a plant or a plant cell; an algal cell, e.g., Botryococcusbraunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorellapyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell(e.g., a yeast cell); an animal cell, tissue, or organ; a cell, tissue,or organ from an invertebrate animal (e.g. fruit fly, cnidarian,echinoderm, nematode, an insect, an arachnid, etc.); a cell, tissue,fluid, or organ from a vertebrate animal (e.g., fish, amphibian,reptile, bird, mammal); a cell, tissue, fluid, or organ from a mammal(e.g., a human; a non-human primate; an ungulate; a feline; a bovine; anovine; a caprine; etc.). Suitable sample sources include nematodes,protozoans, and the like. Suitable sample sources include parasites suchas helminths, malarial parasites, etc.

Suitable sample sources include a cell, tissue, or organism of any ofthe six kingdoms, e.g., Bacteria (e.g., Eubacteria); Archaebacteria;Protista; Fungi; Plantae; and Animalia. Suitable sample sources includeplant-like members of the kingdom Protista, including, but not limitedto, algae (e.g., green algae, red algae, glaucophytes, cyanobacteria);fungus-like members of Protista, e.g., slime molds, water molds, etc ;animal-like members of Protista, e.g., flagellates (e.g., Euglena),amoeboids (e.g., amoeba), sporozoans (e.g, Apicomplexa, Myxozoa,Microsporidia), and ciliates (e.g., Paramecium). Suitable sample sourcesinclude include members of the kingdom Fungi, including, but not limitedto, members of any of the phyla: Basidiomycota (club fungi; e.g.,members of Agaricus, Amanita, Boletus, Cantherellus, etc.); Ascomycota(sac fungi, including, e.g., Saccharomyces); Mycophycophyta (lichens);Zygomycota (conjugation fungi); and Deuteromycota. Suitable samplesources include include members of the kingdom Plantae, including, butnot limited to, members of any of the following divisions: Bryophyta(e.g., mosses), Anthocerotophyta (e.g., hornworts), Hepaticophyta (e.g.,liverworts), Lycophyta (e.g., club mosses), Sphenophyta (e.g.,horsetails), Psilophyta (e.g., whisk ferns), Ophioglossophyta,Pterophyta (e.g., ferns), Cycadophyta, Gingkophyta, Pinophyta,Gnetophyta, and Magnoliophyta (e.g., flowering plants). Suitable samplesources include include members of the kingdom Animalia, including, butnot limited to, members of any of the following phyla: Porifera(sponges); Placozoa; Orthonectida (parasites of marine invertebrates);Rhombozoa; Cnidaria (corals, anemones, jellyfish, sea pens, sea pansies,sea wasps); Ctenophora (comb jellies); Platyhelminthes (flatworms);Nemertina (ribbon worms); Ngathostomulida (jawed worms)p Gastrotricha;Rotifera; Priapulida; Kinorhyncha; Loricifera; Acanthocephala;Entoprocta; Nemotoda; Nematomorpha; Cycliophora; Mollusca (mollusks);Sipuncula (peanut worms); Annelida (segmented worms); Tardigrada (waterbears); Onychophora (velvet worms); Arthropoda (including the subphyla:Chelicerata, Myriapoda, Hexapoda, and Crustacea, where the Cheliceratainclude, e.g., arachnids, Merostomata, and Pycnogonida, where theMyriapoda include, e.g., Chilopoda (centipedes), Diplopoda (millipedes),Paropoda, and Symphyla, where the Hexapoda include insects, and wherethe Crustacea include shrimp, krill, barnacles, etc.; Phoronida;Ectoprocta (moss animals); Brachiopoda; Echinodermata (e.g. starfish,sea daisies, feather stars, sea urchins, sea cucumbers, brittle stars,brittle baskets, etc.); Chaetognatha (arrow worms); Hemichordata (acornworms); and Chordata. Suitable members of Chordata include any member ofthe following subphyla: Urochordata (sea squirts; including Ascidiacea,Thaliacea, and Larvacea); Cephalochordata (lancelets); Myxini (hagfish);and Vertebrata, where members of Vertebrata include, e.g., members ofPetromyzontida (lampreys), Chondrichthyces (cartilaginous fish),Actinopterygii (ray-finned fish), Actinista (coelocanths), Dipnoi(lungfish), Reptilia (reptiles, e.g., snakes, alligators, crocodiles,lizards, etc.), Ayes (birds); and Mammalian (mammals) Suitable plantsinclude any monocotyledon and any dicotyledon.

Suitable sources of a sample include cells, fluid, tissue, or organtaken from an organism; from a particular cell or group of cellsisolated from an organism; etc. For example, where the organism is aplant, suitable sources include xylem, the phloem, the cambium layer,leaves, roots, etc. Where the organism is an animal, suitable sourcesinclude particular tissues (e.g., lung, liver, heart, kidney, brain,spleen, skin, fetal tissue, etc.), or a particular cell type (e.g.,neuronal cells, epithelial cells, endothelial cells, astrocytes,macrophages, glial cells, islet cells, T lymphocytes, B lymphocytes,etc.).

In some cases, the source of the sample is a (or is suspected of being adiseased cell, fluid, tissue, or organ. In some cases, the source of thesample is a normal (non-diseased) cell, fluid, tissue, or organ. In somecases, the source of the sample is a (or is suspected of being apathogen-infected cell, tissue, or organ. For example, the source of asample can be an individual who may or may not be infected—and thesample could be any biological sample (e.g., blood, saliva, biopsy,plasma, serum, bronchoalveolar lavage, sputum, a fecal sample,cerebrospinal fluid, a fine needle aspirate, a swab sample (e.g., abuccal swab, a cervical swab, a nasal swab), interstitial fluid,synovial fluid, nasal discharge, tears, buffy coat, a mucous membranesample, an epithelial cell sample (e.g., epithelial cell scraping),etc.) collected from the individual. In some cases, the sample is acell-free liquid sample. In some cases, the sample is a liquid samplethat can comprise cells. Pathogens include viruses, fungi, helminths,protozoa, malarial parasites, Plasmodium parasites, Toxoplasmaparasites, Schistosoma parasites, and the like. “Helminths” includeroundworms, heartworms, and phytophagous nematodes (Nematoda), flukes(Tematoda), Acanthocephala, and tapeworms (Cestoda). Protozoaninfections include infections from Giardia spp., Trichomonas spp.,African trypanosomiasis, amoebic dysentery, babesiosis, balantidialdysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis.Examples of pathogens such as parasitic/protozoan pathogens include, butare not limited to: Plasmodium falciparum, Plasmodium vivax, Trypanosomacruzi and Toxoplasma gondii. Fungal pathogens include, but are notlimited to: Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis,and Candida albicans. Pathogenic viruses include, e.g., immunodeficiencyvirus (e.g., HIV); influenza virus; dengue; West Nile virus; herpesvirus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A;Hepatitis Virus B; papillomavirus; and the like. Pathogenic viruses caninclude DNA viruses such as: a papovavirus (e.g., human papillomavirus(HPV), polyomavirus); a hepadnavirus (e.g., Hepatitis B Virus (HBV)); aherpesvirus (e.g., herpes simplex virus (HSV), varicella zoster virus(VZV), epstein-barr virus (EBV), cytomegalovirus (CMV), herpeslymphotropic virus, Pityriasis Rosea, kaposi's sarcoma-associatedherpesvirus); an adenovirus (e.g., atadenovirus, aviadenovirus,ichtadenovirus, mastadenovirus, siadenovirus); a poxvirus (e.g.,smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus,pseudocowpox, bovine papular stomatitis virus; tanapox virus, yabamonkey tumor virus; molluscum contagiosum virus (MCV)); a parvovirus(e.g., adeno-associated virus (AAV), Parvovirus B19, human bocavirus,bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; Phycodnaviridae;and the like. Pathogens can include, e.g., DNAviruses [e.g.: apapovavirus (e.g., human papillomavirus (HPV), polyomavirus); ahepadnavirus (e.g., Hepatitis B Virus (HBV)); a herpesvirus (e.g.,herpes simplex virus (HSV), varicella zoster virus (VZV), epstein-barrvirus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus,Pityriasis Rosea, kaposi's sarcoma-associated herpesvirus); anadenovirus (e.g., atadenovirus, aviadenovirus, ichtadenovirus,mastadenovirus, siadenovirus); a poxvirus (e.g., smallpox, vacciniavirus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovinepapular stomatitis virus; tanapox virus, yaba monkey tumor virus;molluscum contagiosum virus (MCV)); a parvovirus (e.g., adeno-associatedvirus (AAV), Parvovirus B19, human bocavirus, bufavirus, human parv4G1); Geminiviridae; Nanoviridae; Phycodnaviridae; and the like],Mycobacterium tuberculosis, Streptococcus agalactiae,methicillin-resistant Staphylococcus aureus, Legionella pneumophila,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans,Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpessimplex virus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, Reovirus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, West Nile virus, Plasmodiumfalciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli,Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei,Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeriatenella, Onchocerca volvulus, Leishmania tropica, Mycobacteriumtuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena,Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoidescorti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae.

Measuring a Detectable Signal

In some cases, a subject method includes a step of measuring (e.g.,measuring a detectable signal produced by Type V CRISPR/Cas effectorprotein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d,Cas12e)-mediated ssDNA cleavage). Because a Type V CRISPR/Cas effectorprotein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d,Cas12e) cleaves non-targeted ssDNA once activated, which occurs when aguide RNA hybridizes with a target DNA in the presence of a Type VCRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a,Cas12b, Cas12c, Cas12d, Cas12e), a detectable signal can be any signalthat is produced when ssDNA is cleaved. For example, in some cases thestep of measuring can include one or more of: gold nanoparticle baseddetection (e.g., see Xu et al., Angew Chem Int Ed Engl.2007;46(19):3468-70; and Xia et al., Proc Natl Acad Sci U S A. 2010 Jun15;107(24):10837-41), fluorescence polarization, colloid phasetransition/dispersion (e.g., Baksh et al., Nature. 2004 Jan8;427(6970):139-41), electrochemical detection, semiconductor-basedsensing (e.g., Rothberg et al., Nature. 2011 Jul 20;475(7356):348-52;e.g., one could use a phosphatase to generate a pH change after ssDNAcleavage reactions, by opening 2′-3′ cyclic phosphates, and by releasinginorganic phosphate into solution), and detection of a labeled detectorssDNA (see elsewhere herein for more details). The readout of suchdetection methods can be any convenient readout. Examples of possiblereadouts include but are not limited to: a measured amount of detectablefluorescent signal; a visual analysis of bands on a gel (e.g., bandsthat represent cleaved product versus uncleaved substrate), a visual orsensor based detection of the presence or absence of a color (i.e.,color detection method), and the presence or absence of (or a particularamount of) an electrical signal.

The measuring can in some cases be quantitative, e.g., in the sense thatthe amount of signal detected can be used to determine the amount oftarget DNA present in the sample. The measuring can in some cases bequalitative, e.g., in the sense that the presence or absence ofdetectable signal can indicate the presence or absence of targeted DNA(e.g., virus, SNP, etc.). In some cases, a detectable signal will not bepresent (e.g., above a given threshold level) unless the targeted DNA(s)(e.g., virus, SNP, etc.) is present above a particular thresholdconcentration. In some cases, the threshold of detection can be titratedby modifying the amount of Type V CRISPR/Cas effector protein (e.g., aCas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e), guideRNA, sample volume, and/or detector ssDNA (if one is used). As such, forexample, as would be understood by one of ordinary skill in the art, anumber of controls can be used if desired in order to set up one or morereactions, each set up to detect a different threshold level of targetDNA, and thus such a series of reactions could be used to determine theamount of target DNA present in a sample (e.g., one could use such aseries of reactions to determine that a target DNA is present in thesample ‘at a concentration of at least X’). Non-limiting examples ofapplications of/uses for the compositions and methods of the disclosureinclude those depicted in FIG. 46. The figure depicts embodiments inwhich nucleic acids of the sample are amplified (denoted as “RPA” inFIG. 46) prior to contact with a Cas12 protein, but the sameapplications/uses (e.g., SNP detection, cancer screening, detection ofbacterial infection, detection of antibiotic resistance, detection ofviral infection, and the like) can apply to embodiments in which noamplification step is included. The compositions and methods of thisdisclosure can be used to detect any DNA target. For example, any virusthat integrates nucleic acid material into the genome can be detectedbecause a subject sample can include cellular genomic DNA—and the guideRNA can be designed to detect integrated nucleotide sequence.

In some cases, a method of the present disclosure can be used todetermine the amount of a target DNA in a sample (e.g., a samplecomprising the target DNA and a plurality of non-target DNAs).Determining the amount of a target DNA in a sample can comprisecomparing the amount of detectable signal generated from a test sampleto the amount of detectable signal generated from a reference sample.Determining the amount of a target DNA in a sample can comprise:measuring the detectable signal to generate a test measurement;measuring a detectable signal produced by a reference sample to generatea reference measurement; and comparing the test measurement to thereference measurement to determine an amount of target DNA present inthe sample.

For example, in some cases, a method of the present disclosure fordetermining the amount of a target DNA in a sample comprises: a)contacting the sample (e.g., a sample comprising the target DNA and aplurality of non-target DNAs) with: (i) a guide RNA that hybridizes withthe target DNA, (ii) a Type V CRISPR/Cas effector protein (e.g., a Cas12protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) that cleavesRNAs present in the sample, and (iii) a detector ssDNA; b) measuring adetectable signal produced by Type V CRISPR/Cas effector protein (e.g.,a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e)-mediatedssDNA cleavage (e.g., cleavage of the detector ssDNA), generating a testmeasurement; c) measuring a detectable signal produced by a referencesample to generate a reference measurement; and d) comparing the testmeasurement to the reference measurement to determine an amount oftarget DNA present in the sample.

As another example, in some cases, a method of the present disclosurefor determining the amount of a target DNA in a sample comprises: a)contacting the sample (e.g., a sample comprising the target DNA and aplurality of non-target DNAs) with: i) a precursor guide RNA arraycomprising two or more guide RNAs each of which has a different guidesequence; (ii) a Type V CRISPR/Cas effector protein (e.g., a Cas12protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) that cleaves theprecursor guide RNA array into individual guide RNAs, and also cleavesRNAs of the sample; and (iii) a detector ssDNA; b) measuring adetectable signal produced by Type V CRISPR/Cas effector protein (e.g.,a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e)-mediatedssDNA cleavage (e.g., cleavage of the detector ssDNA), generating a testmeasurement; c) measuring a detectable signal produced by each of two ormore reference samples to generate two or more reference measurements;and d) comparing the test measurement to the reference measurements todetermine an amount of target DNA present in the sample.

Amplification of Nucleic Acids in the Sample

In some embodiments, sensitivity of a subject composition and/or method(e.g., for detecting the presence of a target DNA, such as viral DNA ora SNP, in cellular genomic DNA) can be increased by coupling detectionwith nucleic acid amplification. In some cases, the nucleic acids in asample are amplified prior to contact with a type V CRISPR/Cas effectorprotein (e.g., a Cas12 protein) that cleaved ssDNA (e.g., amplificationof nucleic acids in the sample can begin prior to contact with a type VCRISPR/Cas effector protein). In some cases, the nucleic acids in asample are amplified simultaneous with contact with a type V CRISPR/Caseffector protein (e.g., a Cas12 protein). For example, in some cases asubject method includes amplifying nucleic acids of a sample (e.g., bycontacting the sample with amplification components) prior to contactingthe amplified sample with a type V CRISPR/Cas effector protein (e.g., aCas12 protein). In some cases a subject method includes contacting asample with amplification components at the same time (simultaneouswith) that the sample is contacted with a type V CRISPR/Cas effectorprotein (e.g., a Cas12 protein). If all components are addedsimultaneously (amplification components and detection components suchas a type V CRISPR/Cas effector protein, e.g., a Cas12 protein, a guideRNA, and a detector DNA), it is possible that the trans-cleavageactivity of the type V CRISPR/Cas effector protein (e.g., a Cas12protein), will begin to degrade the nucleic acids of the sample at thesame time the nucleic acids are undergoing amplification. However, evenif this is the case, amplifying and detecting simultaneously can stillincrease sensitivity compared to performing the method withoutamplification.

In some cases specific sequences (e.g., sequences of a virus, sequencesthat include a SNP of interest) are amplified from the sample, e.g.,using primers. As such, a sequence to which the guide RNA will hybridizecan be amplified in order to increase sensitivity of a subject detectionmethod—this could achieve biased amplification of a desired sequence inorder to increase the number of copies of the sequence of interestpresent in the sample relative to other sequences present in the sample.As one illustrative example, if a subject method is being used todetermine whether a given sample includes a particular virus (or aparticular SNP), a desired region of viral sequence (or non-viralgenomic sequence) can be amplified, and the region amplified willinclude the sequence that would hybridize to the guide RNA if the viralsequence (or SNP) were in fact present in the sample.

As noted, in some cases the nucleic acids are amplified (e.g., bycontact with amplification components) prior to contacting the amplifiednucleic acids with a type V CRISPR/Cas effector protein (e.g., a Cas12protein). In some cases, amplification occurs for 10 seconds or more,(e.g., 30 seconds or more, 45 seconds or more, 1 minute or more, 2minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes ormore, 7.5 minutes or more, 10 minutes or more, etc.) prior to contactwith an active type V CRISPR/Cas effector protein (e.g., a Cas12protein). In some cases, amplification occurs for 2 minutes or more(e.g., 3 minutes or more, 4 minutes or more, 5 minutes or more, 7.5minutes or more, 10 minutes or more, etc.) prior to contact with anactive type V CRISPR/Cas effector protein (e.g., a Cas12 protein). Insome cases, amplification occurs for a period of time in a range of from10 seconds to 60 minutes (e.g., 10 seconds to 40 minutes, 10 seconds to30 minutes, 10 seconds to 20 minutes, 10 seconds to 15 minutes, 10seconds to 10 minutes, 10 seconds to 5 minutes, 30 seconds to 40minutes, 30 seconds to 30 minutes, 30 seconds to 20 minutes, 30 secondsto 15 minutes, 30 seconds to 10 minutes, 30 seconds to 5 minutes, 1minute to 40 minutes, 1 minute to 30 minutes, 1 minute to 20 minutes, 1minute to 15 minutes, 1 minute to 10 minutes, 1 minute to 5 minutes, 2minutes to 40 minutes, 2 minutes to 30 minutes, 2 minutes to 20 minutes,2 minutes to 15 minutes, 2 minutes to 10 minutes, 2 minutes to 5minutes, 5 minutes to 40 minutes, 5 minutes to 30 minutes, 5 minutes to20 minutes, 5 minutes to 15 minutes, or 5 minutes to 10 minutes). Insome cases, amplification occurs for a period of time in a range of from5 minutes to 15 minutes. In some cases, amplification occurs for aperiod of time in a range of from 7 minutes to 12 minutes.

In some cases, a sample is contacted with amplification components atthe same time as contact with a type V CRISPR/Cas effector protein(e.g., a Cas12 protein). In some such cases, the type V CRISPR/Caseffector protein in inactive at the time of contact and is activatedonce nucleic acids in the sample have been amplified.

Various amplification methods and components will be known to one ofordinary skill in the art and any convenient method can be used (see,e.g., Zanoli and Spoto, Biosensors (Basel). 2013 Mar; 3(1): 18-43; Gilland Ghaemi, Nucleosides, Nucleotides, and Nucleic Acids, 2008, 27:224-243; Craw and Balachandrana, Lab Chip, 2012, 12, 2469-2486; whichare herein incorporated by reference in their entirety). Nucleic acidamplification can comprise polymerase chain reaction (PCR), reversetranscription PCR (RT-PCR), quantitative PCR (qPCR), reversetranscription qPCR (RT-qPCR), nested PCR, multiplex PCR, asymmetric PCR,touchdown PCR, random primer PCR, hemi-nested PCR, polymerase cyclingassembly (PCA), colony PCR, ligase chain reaction (LCR), digital PCR,methylation specific-PCR (MSP),co-amplification at lower denaturationtemperature-PCR (COLD-PCR), allele-specific PCR, intersequence-specificPCR (ISS-PCR), whole genome amplification (WGA), inverse PCR, andthermal asymmetric interlaced PCR (TAIL-PCR).

In some cases the amplification is isothermal amplification. The term“isothermal amplification” indicates a method of nucleic acid (e.g.,DNA) amplification (e.g., using enzymatic chain reaction) that can use asingle temperature incubation thereby obviating the need for a thermalcycler. Isothermal amplification is a form of nucleic acid amplificationwhich does not rely on the thermal denaturation of the target nucleicacid during the amplification reaction and hence may not requiremultiple rapid changes in temperature. Isothermal nucleic acidamplification methods can therefore be carried out inside or outside ofa laboratory environment. By combining with a reverse transcriptionstep, these amplification methods can be used to isothermally amplifyRNA.

Examples of isothermal amplification methods include but are not limitedto: loop-mediated isothermal Amplification (LAMP), helicase-dependentAmplification (HDA), recombinase polymerase amplification (RPA), stranddisplacement amplification (SDA), nucleic acid sequence-basedamplification (NASBA), transcription mediated amplification (TMA),nicking enzyme amplification reaction (NEAR), rolling circleamplification (RCA), multiple displacement amplification (MDA),Ramification (RAM), circular helicase-dependent amplification (cHDA),single primer isothermal amplification (SPIA), signal mediatedamplification of RNA technology (SMART), self-sustained sequencereplication (3SR), genome exponential amplification reaction (GEAR) andisothermal multiple displacement amplification (IMDA).

In some cases, the amplification is recombinase polymerase amplification(RPA) (see, e.g., U.S. Pat. Nos. 8,030,000; 8,426,134; 8,945,845;9,309,502; and 9,663,820, which are hereby incorporated by reference intheir entirety). Recombinase polymerase amplification (RPA) uses twoopposing primers (much like PCR) and employs three enzymes—arecombinase, a single-stranded DNA-binding protein (SSB) and astrand-displacing polymerase. The recombinase pairs oligonucleotideprimers with homologous sequence in duplex DNA, SSB binds to displacedstrands of DNA to prevent the primers from being displaced, and thestrand displacing polymerase begins DNA synthesis where the primer hasbound to the target DNA. Adding a reverse transcriptase enzyme to an RPAreaction can facilitate detection RNA as well as DNA, without the needfor a separate step to produce cDNA. One example of components for anRPA reaction is as follows (see, e.g., U.S. Pat. Nos. 8,030,000;8,426,134; 8,945,845; 9,309,502; 9,663,820): 50 mM Tris pH 8.4, 80 mMPotassium actetate, 10 mM Magnesium acetate, 2 mM DTT, 5% PEG compound(Carbowax-20M), 3 mM ATP, 30 mM Phosphocreatine, 100 ng/μ1 creatinekinase, 420 ng/μ1 gp32, 140 ng/μ1 UvsX, 35 ng/μl UvsY, 2000M dNTPs, 300nM each oligonucleotide, 35 ng/μ1 Bsu polymerase, and a nucleicacid-containing sample).

In a transcription mediated amplification (TMA), an RNA polymerase isused to make RNA from a promoter engineered in the primer region, andthen a reverse transcriptase synthesizes cDNA from the primer. A thirdenzyme, e.g., Rnase H can then be used to degrade the RNA target fromcDNA without the heat-denatured step. This amplification technique issimilar to Self-Sustained Sequence Replication (3SR) and Nucleic AcidSequence Based Amplification (NASBA), but varies in the enzymesemployed. For another example, helicase-dependent amplification (HDA)utilizes a thermostable helicase (Tte-UvrD) rather than heat to unwinddsDNA to create single-strands that are then available for hybridizationand extension of primers by polymerase. For yet another example, a loopmediated amplification (LAMP) employs a thermostable polymerase withstrand displacement capabilities and a set of four or more specificdesigned primers. Each primer is designed to have hairpin ends that,once displaced, snap into a hairpin to facilitate self-priming andfurther polymerase extension. In a LAMP reaction, though the reactionproceeds under isothermal conditions, an initial heat denaturation stepis required for double-stranded targets. In addition, amplificationyields a ladder pattern of various length products. For yet anotherexample, a strand displacement amplification (SDA) combines the abilityof a restriction endonuclease to nick the unmodified strand of itstarget DNA and an exonuclease-deficient DNA polymerase to extend the 3′end at the nick and displace the downstream DNA strand.

Detector DNA

In some cases, a subject method includes contacting a sample (e.g., asample comprising a target DNA and a plurality of non-target ssDNAs)with: i) a Type V CRISPR/Cas effector protein (e.g., a Cas12 proteinsuch as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e); ii) a guide RNA (orprecursor guide RNA array); and iii) a detector DNA that is singlestranded and does not hybridize with the guide sequence of the guideRNA. For example, in some cases, a subject method includes contacting asample with a labeled single stranded detector DNA (detector ssDNA) thatincludes a fluorescence-emitting dye pair; the Type V CRISPR/Caseffector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c,Cas12d, Cas12e) cleaves the labeled detector ssDNA after it is activated(by binding to the guide RNA in the context of the guide RNA hybridizingto a target DNA); and the detectable signal that is measured is producedby the fluorescence-emitting dye pair. For example, in some cases, asubject method includes contacting a sample with a labeled detectorssDNA comprising a fluorescence resonance energy transfer (FRET) pair ora quencher/fluor pair, or both. In some cases, a subject method includescontacting a sample with a labeled detector ssDNA comprising a FRETpair. In some cases, a subject method includes contacting a sample witha labeled detector ssDNA comprising a fluor/quencher pair.

Fluorescence-emitting dye pairs comprise a FRET pair or a quencher/fluorpair. In both cases of a FRET pair and a quencher/fluor pair, theemission spectrum of one of the dyes overlaps a region of the absorptionspectrum of the other dye in the pair. As used herein, the term“fluorescence-emitting dye pair” is a generic term used to encompassboth a “fluorescence resonance energy transfer (FRET) pair” and a“quencher/fluor pair,” both of which terms are discussed in more detailbelow. The term “fluorescence-emitting dye pair” is used interchangeablywith the phrase “a FRET pair and/or a quencher/fluor pair.”

In some cases (e.g., when the detector ssDNA includes a FRET pair) thelabeled detector ssDNA produces an amount of detectable signal prior tobeing cleaved, and the amount of detectable signal that is measured isreduced when the labeled detector ssDNA is cleaved. In some cases, thelabeled detector ssDNA produces a first detectable signal prior to beingcleaved (e.g., from a FRET pair) and a second detectable signal when thelabeled detector ssDNA is cleaved (e.g., from a quencher/fluor pair). Assuch, in some cases, the labeled detector ssDNA comprises a FRET pairand a quencher/fluor pair.

In some cases, the labeled detector ssDNA comprises a FRET pair. FRET isa process by which radiationless transfer of energy occurs from anexcited state fluorophore to a second chromophore in close proximity Therange over which the energy transfer can take place is limited toapproximately 10 nanometers (100 angstroms), and the efficiency oftransfer is extremely sensitive to the separation distance betweenfluorophores. Thus, as used herein, the term “FRET” (“fluorescenceresonance energy transfer”; also known as “Förster resonance energytransfer”) refers to a physical phenomenon involving a donor fluorophoreand a matching acceptor fluorophore selected so that the emissionspectrum of the donor overlaps the excitation spectrum of the acceptor,and further selected so that when donor and acceptor are in closeproximity (usually 10 nm or less) to one another, excitation of thedonor will cause excitation of and emission from the acceptor, as someof the energy passes from donor to acceptor via a quantum couplingeffect. Thus, a FRET signal serves as a proximity gauge of the donor andacceptor; only when they are in close proximity to one another is asignal generated. The FRET donor moiety (e.g., donor fluorophore) andFRET acceptor moiety (e.g., acceptor fluorophore) are collectivelyreferred to herein as a “FRET pair”.

The donor-acceptor pair (a FRET donor moiety and a FRET acceptor moiety)is referred to herein as a “FRET pair” or a “signal FRET pair.” Thus, insome cases, a subject labeled detector ssDNA includes two signalpartners (a signal pair), when one signal partner is a FRET donor moietyand the other signal partner is a FRET acceptor moiety. A subjectlabeled detector ssDNA that includes such a FRET pair (a FRET donormoiety and a FRET acceptor moiety) will thus exhibit a detectable signal(a FRET signal) when the signal partners are in close proximity (e.g.,while on the same RNA molecule), but the signal will be reduced (orabsent) when the partners are separated (e.g., after cleavage of the RNAmolecule by a Type V CRISPR/Cas effector protein (e.g., a Cas12 proteinsuch as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e)).

FRET donor and acceptor moieties (FRET pairs) will be known to one ofordinary skill in the art and any convenient FRET pair (e.g., anyconvenient donor and acceptor moiety pair) can be used. Examples ofsuitable FRET pairs include but are not limited to those presented inTable 1. See also: Bajar et al. Sensors (Basel). 2016 Sep. 14; 16(9);and Abraham et al. PLoS One. 2015 Aug. 3; 10(8):e0134436.

TABLE 1 Examples of FRET pairs (donor and acceptor FRET moieties) DonorAcceptor Tryptophan Dansyl IAEDANS (1) DDPM (2) BFP DsRFP DansylFluorescein isothiocyanate (FITC) Dansyl Octadecylrhodamine Cyanfluorescent protein (CFP) Green fluorescent protein (GFP) CF (3) TexasRed Fluorescein Tetramethylrhodamine Cy3 Cy5 GFP Yellow fluorescentprotein (YFP) BODIPY FL (4) BODIPY FL (4) Rhodamine 110 Cy3 Rhodamine 6GMalachite Green FITC Eosin Thiosemicarbazide B-Phycoerythrin Cy5 Cy5Cy5.5 (1) 5-(2-iodoacetylaminoethyl)aminonaphthalene-1-sulfonic acid (2)N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (3) carboxyfluoresceinsuccinimidyl ester (4) 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

In some cases, a detectable signal is produced when the labeled detectorssDNA is cleaved (e.g., in some cases, the labeled detector ssDNAcomprises a quencher/fluor pair). One signal partner of a signalquenching pair produces a detectable signal and the other signal partneris a quencher moiety that quenches the detectable signal of the firstsignal partner (i.e., the quencher moiety quenches the signal of thesignal moiety such that the signal from the signal moiety is reduced(quenched) when the signal partners are in proximity to one another,e.g., when the signal partners of the signal pair are in closeproximity).

For example, in some cases, an amount of detectable signal increaseswhen the labeled detector ssDNA is cleaved. For example, in some cases,the signal exhibited by one signal partner (a signal moiety) is quenchedby the other signal partner (a quencher signal moiety), e.g., when bothare present on the same ssDNA molecule prior to cleavage by a Type VCRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a,Cas12b, Cas12c, Cas12d, Cas12e). Such a signal pair is referred toherein as a “quencher/fluor pair”, “quenching pair”, or “signalquenching pair.” For example, in some cases, one signal partner (e.g.,the first signal partner) is a signal moiety that produces a detectablesignal that is quenched by the second signal partner (e.g., a quenchermoiety). The signal partners of such a quencher/fluor pair will thusproduce a detectable signal when the partners are separated (e.g., aftercleavage of the detector ssDNA by a Type V CRISPR/Cas effector protein(e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e)),but the signal will be quenched when the partners are in close proximity(e.g., prior to cleavage of the detector ssDNA by a Type V CRISPR/Caseffector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c,Cas12d, Cas12e)).

A quencher moiety can quench a signal from the signal moiety (e.g.,prior to cleave of the detector ssDNA by a Type V CRISPR/Cas effectorprotein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d,Cas12e)) to various degrees. In some cases, a quencher moiety quenchesthe signal from the signal moiety where the signal detected in thepresence of the quencher moiety (when the signal partners are inproximity to one another) is 95% or less of the signal detected in theabsence of the quencher moiety (when the signal partners are separated).For example, in some cases, the signal detected in the presence of thequencher moiety can be 90% or less, 80% or less, 70% or less, 60% orless, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less,10% or less, or 5% or less of the signal detected in the absence of thequencher moiety. In some cases, no signal (e.g., above background) isdetected in the presence of the quencher moiety.

In some cases, the signal detected in the absence of the quencher moiety(when the signal partners are separated) is at least 1.2 fold greater(e.g., at least 1.3fold, at least 1.5 fold, at least 1.7 fold, at least2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least4 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 20fold, or at least 50 fold greater) than the signal detected in thepresence of the quencher moiety (when the signal partners are inproximity to one another).

In some cases, the signal moiety is a fluorescent label. In some suchcases, the quencher moiety quenches the signal (the light signal) fromthe fluorescent label (e.g., by absorbing energy in the emission spectraof the label). Thus, when the quencher moiety is not in proximity withthe signal moiety, the emission (the signal) from the fluorescent labelis detectable because the signal is not absorbed by the quencher moiety.Any convenient donor acceptor pair (signal moiety/quencher moiety pair)can be used and many suitable pairs are known in the art.

In some cases the quencher moiety absorbs energy from the signal moiety(also referred to herein as a “detectable label”) and then emits asignal (e.g., light at a different wavelength). Thus, in some cases, thequencher moiety is itself a signal moiety (e.g., a signal moiety can be6-carboxyfluorescein while the quencher moiety can be6-carboxy-tetramethylrhodamine), and in some such cases, the pair couldalso be a FRET pair. In some cases, a quencher moiety is a darkquencher. A dark quencher can absorb excitation energy and dissipate theenergy in a different way (e.g., as heat). Thus, a dark quencher hasminimal to no fluorescence of its own (does not emit fluorescence).Examples of dark quenchers are further described in U.S. Pat. Nos.8,822,673 and 8,586,718; U.S. patent publications 20140378330,20140349295, and 20140194611; and international patent applications:WO200142505 and WO200186001, all if which are hereby incorporated byreference in their entirety.

Examples of fluorescent labels include, but are not limited to: an AlexaFluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488,ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550,ATTO 565, ATTO Rho3B, ATTO Rhol1, ATTO Rhol12, ATTO Thio12, ATTO Rho101,ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3,Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye, a Sulfo Cy dye,a Seta dye, an IRIS Dye, a SeTau dye, an SRfluor dye, a Square dye,fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), TexasRed, Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, quantumdots, and a tethered fluorescent protein.

In some cases, a detectable label is a fluorescent label selected from:an Alexa Fluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465,ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542,ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol1, ATTO Rhol12, ATTO Thio12,ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTORho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665,ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye(e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye,a Sulfo Cy dye, a Seta dye, an IRIS Dye, a SeTau dye, an SRfluor dye, aSquare dye, fluorescein (FITC), tetramethylrhodamine (TRITC), Texas Red,Oregon Green, Pacific Blue, Pacific Green, and Pacific Orange.

In some cases, a detectable label is a fluorescent label selected from:an Alexa Fluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465,ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542,ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol1, ATTO Rhol12, ATTO Thio12,ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTORho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665,ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye(e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye,a Sulfo Cy dye, a Seta dye, an IRIS Dye, a SeTau dye, an SRfluor dye, aSquare dye, fluorescein (FITC), tetramethylrhodamine (TRITC), Texas Red,Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, a quantumdot, and a tethered fluorescent protein.

Examples of ATTO dyes include, but are not limited to: ATTO 390, ATTO425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTORho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol1, ATTO Rho12,ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12,ATTO 665, ATTO 680, ATTO 700, ATTO 725, and ATTO 740.

Examples of AlexaFluor dyes include, but are not limited to: AlexaFluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, AlexaFluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, AlexaFluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, AlexaFluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, AlexaFluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Alexa Fluor® 790, andthe like.

Examples of quencher moieties include, but are not limited to: a darkquencher, a Black Hole Quencher® (BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2,BHQ-3), a Q×1 quencher, an ATTO quencher (e.g., ATTO 540Q, ATTO 580Q,and ATTO 612Q), dimethylaminoazobenzenesulfonic acid (Dabsyl), IowaBlack RQ, Iowa Black FQ, IRDye QC-1, a QSY dye (e.g., QSY 7, QSY 9, QSY21), AbsoluteQuencher, Eclipse, and metal clusters such as goldnanoparticles, and the like.

In some cases, a quencher moiety is selected from: a dark quencher, aBlack Hole Quencher® (BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3), a Qxlquencher, an ATTO quencher (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q),dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa BlackFQ, IRDye QC-1, a QSY dye (e.g., QSY 7, QSY 9, QSY 21),AbsoluteQuencher, Eclipse, and a metal cluster.

Examples of an ATTO quencher include, but are not limited to: ATTO 540Q,ATTO 580Q, and ATTO 612Q. Examples of a Black Hole Quencher® (BHQ®)include, but are not limited to: BHQ-0 (493 nm), BHQ-1 (534 nm), BHQ-2(579 nm) and BHQ-3 (672 nm).

For examples of some detectable labels (e.g., fluorescent dyes) and/orquencher moieties, see, e.g., Bao et al., Annu Rev Biomed Eng.2009;11:25-47; as well as U.S. Pat. Nos. 8,822,673 and 8,586,718; U.S.patent publications 20140378330, 20140349295,20140194611,20130323851,20130224871,20110223677,20110190486,20110172420,20060179585 and 20030003486; and international patent applications:WO200142505 and WO200186001, all of which are hereby incorporated byreference in their entirety.

In some cases, cleavage of a labeled detector ssDNA can be detected bymeasuring a colorimetric read-out. For example, the liberation of afluorophore (e.g., liberation from a FRET pair, liberation from aquencher/fluor pair, and the like) can result in a wavelength shift (andthus color shift) of a detectable signal. Thus, in some cases, cleavageof a subject labeled detector ssDNA can be detected by a color-shift.Such a shift can be expressed as a loss of an amount of signal of onecolor (wavelength), a gain in the amount of another color, a change inthe ration of one color to another, and the like.

Type V CRISPR/Cas Effector Proteins

Type V CRISPR/Cas effector proteins are a subtype of Class 2 CRISPR/Caseffector proteins. For examples of type V CRISPR/Cas systems and theireffector proteins (e.g., Cas12 family proteins such as Cas12a), see,e.g., Shmakov et al., Nat Rev Microbiol. 2017 Mar; 15(3):169-182:“Diversity and evolution of class 2 CRISPR-Cas systems.” Examplesinclude, but are not limited to: Cas12 family (Cas12a, Cas12b, Cas12c),C2c4, C2c8, C2c5, C2c10, and C2c9; as well as CasX (Cas12e) and CasY(Cas12d). Also see, e.g., Koonin et al., Curr Opin Microbiol. 2017 Jun;37:67-78: “Diversity, classification and evolution of CRISPR-Cassystems.”

As such in some cases, a subject type V CRISPR/Cas effector protein is aCas12 protein (e.g., Cas12a, Cas12b, Cas12c). In some cases, a subjecttype V CRISPR/Cas effector protein is a Cas12 protein such as Cas12a,Cas12b, Cas12c, Cas12d, Cas12e, Cas12d, or Cas12e. In some cases, asubject type V CRISPR/Cas effector protein is a Cas12a protein. In somecases, a subject type V CRISPR/Cas effector protein is a Cas12b protein.In some cases, a subject type V CRISPR/Cas effector protein is a Cas12cprotein. In some cases, a subject type V CRISPR/Cas effector protein isa Cas12d protein. In some cases, a subject type V CRISPR/Cas effectorprotein is a Cas12e protein. In some cases, a subject type V CRISPR/Caseffector protein is protein selected from: Cas12 (e.g., Cas12a, Cas12b,Cas12c, Cas12d, Cas12e), C2c4, C2c8, C2c5, C2c10, and C2c9. In somecases, a subject type V CRISPR/Cas effector protein is protein selectedfrom: C2c4, C2c8, C2c5, C2c10, and C2c9. In some cases, a subject type VCRISPR/Cas effector protein is protein selected from: C2c4, C2c8, andC2c5. In some cases, a subject type V CRISPR/Cas effector protein isprotein selected from: C2c10 and C2c9.

In some cases, the subject type V CRISPR/Cas effector protein is anaturally-occurring protein (e.g., naturally occurs in prokaryoticcells). In other cases, the Type V CRISPR/Cas effector protein is not anaturally-occurring polypeptide (e.g., the effector protein is a variantprotein, a chimeric protein, includes a fusion partner, and the like).Examples of naturally occurring Type V CRISPR/Cas effector proteinsinclude, but are not limited to, those depicted in FIG. 1. Any Type VCRISPR/Cas effector protein can be suitable for the compositions (e.g.,nucleic acids, kits, etc.) and methods of the present disclosure (e.g.,as long as the Type V CRISPR/Cas effector protein forms a complex with aguide RNA and exhibits ssDNA cleavage activity of non-target ssDNAs onceit is activated (by hybridization of and associated guide RNA to itstarget DNA).

In some cases, a type V CRISPR/Cas effector protein comprises an aminoacid sequence having 20% or more sequence identity (e.g., 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a Cas12 protein (e.g., Cas12a, Cas12b,Cas12c) (e.g., a Cas12 protein depicted in FIG. 1). For example, in somecases a type V CRISPR/Cas effector protein comprises an amino acidsequence having 50% or more sequence identity (e.g., 60% or more, 70% ormore, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more,98% or more, 99% or more, or 100% sequence identity) with a Cas12protein (e.g., Cas12a, Cas12b, Cas12c) (e.g., a Cas12 protein depictedin FIG. 1). In some cases a type V CRISPR/Cas effector protein comprisesan amino acid sequence having 80% or more sequence identity (e.g., 85%or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with a Cas12 protein (e.g., Cas12a,Cas12b, Cas12c) (e.g., a Cas12 protein depicted in FIG. 1). In somecases a type V CRISPR/Cas effector protein comprises an amino acidsequence having 90% or more sequence identity (e.g., 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with a Cas12protein (e.g., Cas12a, Cas12b, Cas12c) (e.g., a Cas12 protein depictedin FIG. 1). In some cases a type V CRISPR/Cas effector protein comprisesa Cas12 amino acid sequence (e.g., Cas12a, Cas12b, Cas12c) depicted inFIG. 1.

In some cases, a type V CRISPR/Cas effector protein comprises an aminoacid sequence having 20% or more sequence identity (e.g., 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a Cas12a protein (e.g., a Cas12a proteindepicted in FIG. 1). For example, in some cases a type V CRISPR/Caseffector protein comprises an amino acid sequence having 50% or moresequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a Cas12a protein (e.g., a Cas12a proteindepicted in FIG. 1). In some cases a type V CRISPR/Cas effector proteincomprises an amino acid sequence having 80% or more sequence identity(e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more,99% or more, or 100% sequence identity) with a Cas12a protein (e.g., aCas12a protein depicted in FIG. 1). In some cases a type V CRISPR/Caseffector protein comprises an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with a Cas12a protein (e.g., a Cas12aprotein depicted in FIG. 1). In some cases a type V CRISPR/Cas effectorprotein comprises a Cas12a amino acid sequence depicted in FIG. 1.

In some cases, a suitable type V CRISPR/Cas effector protein comprisesan amino acid sequence having 20% or more sequence identity (e.g., 30%or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% ormore, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more,99% or more, or 100% sequence identity) with the Lachnospiraceaebacterium ND2006 Cas12a protein amino acid sequence depicted in FIG. 1.In some cases, a suitable type V CRISPR/Cas effector protein comprisesan amino acid sequence having 20% or more sequence identity (e.g., 30%or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% ormore, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more,99% or more, or 100% sequence identity) with the Acidaminococcus spBV3L6Cas12a protein amino acid sequence depicted in FIG. 1. In some cases, asuitable type V CRISPR/Cas effector protein comprises an amino acidsequence having 20% or more sequence identity (e.g., 30% or more, 40% ormore, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more,90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%sequence identity) with the Francisella novicida U112 Cas12a proteinamino acid sequence depicted in FIG. 1. In some cases, a suitable type VCRISPR/Cas effector protein comprises an amino acid sequence having 20%or more sequence identity (e.g., 30% or more, 40% or more, 50% or more,60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100% sequence identity)with the Porphyromonas macacae Cas12a protein amino acid sequencedepicted in FIG. 1. In some cases, a suitable type V CRISPR/Cas effectorprotein comprises an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theMoraxella bovoculi 237 Cas12a protein amino acid sequence depicted inFIG. 1. In some cases, a suitable type V CRISPR/Cas effector proteincomprises an amino acid sequence having 20% or more sequence identity(e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100% sequence identity) with the Moraxellabovoculi AAX08_00205 Cas12a protein amino acid sequence depicted inFIG. 1. In some cases, a suitable type V CRISPR/Cas effector proteincomprises an amino acid sequence having 20% or more sequence identity(e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100% sequence identity) with the Moraxellabovoculi AAX11_00205 Cas12a protein amino acid sequence depicted inFIG. 1. In some cases, a suitable type V CRISPR/Cas effector proteincomprises an amino acid sequence having 20% or more sequence identity(e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100% sequence identity) with the Thiomicrospirasp.XS5 Cas12a protein amino acid sequence depicted in FIG. 1. In somecases, a suitable type V CRISPR/Cas effector protein comprises an aminoacid sequence having 20% or more sequence identity (e.g., 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with the Butyrivibrio sp. NC3005 Cas12aprotein amino acid sequence depicted in FIG. 1. In some cases, asuitable type V CRISPR/Cas effector protein comprises an amino acidsequence having 20% or more sequence identity (e.g., 30% or more, 40% ormore, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more,90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%sequence identity) with the AACCas12b amino acid sequence depicted inFIG. 1.

In some cases, a type V CRISPR/Cas effector protein comprises an aminoacid sequence having 20% or more sequence identity (e.g., 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a Cas12b protein (e.g., a Cas12b proteindepicted in FIG. 1). For example, in some cases a type V CRISPR/Caseffector protein comprises an amino acid sequence having 50% or moresequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a Cas12b protein (e.g., a Cas12b proteindepicted in FIG. 1). In some cases a type V CRISPR/Cas effector proteincomprises an amino acid sequence having 80% or more sequence identity(e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more,99% or more, or 100% sequence identity) with a Cas12b protein (e.g., aCas12b protein depicted in FIG. 1). In some cases a type V CRISPR/Caseffector protein comprises an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with a Cas12b protein (e.g., a Cas12bprotein depicted in FIG. 1). In some cases a type V CRISPR/Cas effectorprotein comprises a Cas12b amino acid sequence depicted in FIG. 1.

In some cases, a type V CRISPR/Cas effector protein comprises an aminoacid sequence having 20% or more sequence identity (e.g., 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a Cas12, C2c4, C2c8, C2c5, C2c10, orC2c9 protein. For example, in some cases a type V CRISPR/Cas effectorprotein comprises an amino acid sequence having 50% or more sequenceidentity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90%or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%sequence identity) with a Cas12, C2c4, C2c8, C2c5, C2c10, or C2c9protein. In some cases a type V CRISPR/Cas effector protein comprises anamino acid sequence having 80% or more sequence identity (e.g., 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a Cas12, C2c4, C2c8, C2c5, C2c10, orC2c9 protein. In some cases a type V CRISPR/Cas effector proteincomprises an amino acid sequence having 90% or more sequence identity(e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%sequence identity) with a Cas12, C2c4, C2c8, C2c5, C2c10, or C2c9protein. In some cases a type V CRISPR/Cas effector protein comprises aCas12, C2c4, C2c8, C2c5, C2c10, or C2c9 amino acid sequence.

In some cases, a type V CRISPR/Cas effector protein comprises an aminoacid sequence having 20% or more sequence identity (e.g., 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a Cas12, C2c4, C2c8, or C2c5protein. Forexample, in some cases a type V CRISPR/Cas effector protein comprises anamino acid sequence having 50% or more sequence identity (e.g., 60% ormore, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) with aCas12, C2c4, C2c8, or C2c5protein. In some cases a type V CRISPR/Caseffector protein comprises an amino acid sequence having 80% or moresequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with a Cas12,C2c4, C2c8, or C2c5protein. In some cases a type V CRISPR/Cas effectorprotein comprises an amino acid sequence having 90% or more sequenceidentity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or100% sequence identity) with a Cas12, C2c4, C2c8, or C2c5protein. Insome cases a type V CRISPR/Cas effector protein comprises a Cas12, C2c4,C2c8, or C2c5amino acid sequence.

In some cases, a type V CRISPR/Cas effector protein comprises an aminoacid sequence having 20% or more sequence identity (e.g., 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a C2c4, C2c8, or C2c5protein. Forexample, in some cases a type V CRISPR/Cas effector protein comprises anamino acid sequence having 50% or more sequence identity (e.g., 60% ormore, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) with aC2c4, C2c8, or C2c5protein. In some cases a type V CRISPR/Cas effectorprotein comprises an amino acid sequence having 80% or more sequenceidentity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98%or more, 99% or more, or 100% sequence identity) with a C2c4, C2c8, orC2c5protein. In some cases a type V CRISPR/Cas effector proteincomprises an amino acid sequence having 90% or more sequence identity(e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%sequence identity) with a C2c4, C2c8, or C2c5protein. In some cases atype V CRISPR/Cas effector protein comprises a C2c4, C2c8, or C2c5aminoacid sequence.

In some cases, a type V CRISPR/Cas effector protein comprises an aminoacid sequence having 20% or more sequence identity (e.g., 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a Cas12, C2c10, or C2c9protein. Forexample, in some cases a type V CRISPR/Cas effector protein comprises anamino acid sequence having 50% or more sequence identity (e.g., 60% ormore, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) with aCas12, C2c10, or C2c9protein. In some cases a type V CRISPR/Cas effectorprotein comprises an amino acid sequence having 80% or more sequenceidentity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98%or more, 99% or more, or 100% sequence identity) with a Cas12, C2c10, orC2c9protein. In some cases a type V CRISPR/Cas effector proteincomprises an amino acid sequence having 90% or more sequence identity(e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%sequence identity) with a Cas12, C2c10, or C2c9protein. In some cases atype V CRISPR/Cas effector protein comprises a Cas12, C2c10, orC2c9amino acid sequence.

In some cases, a type V CRISPR/Cas effector protein comprises an aminoacid sequence having 20% or more sequence identity (e.g., 30% or more,40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with a C2c10 or C2c9protein. For example, insome cases a type V CRISPR/Cas effector protein comprises an amino acidsequence having 50% or more sequence identity (e.g., 60% or more, 70% ormore, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more,98% or more, 99% or more, or 100% sequence identity) with a C2c10 orC2c9protein. In some cases a type V CRISPR/Cas effector proteincomprises an amino acid sequence having 80% or more sequence identity(e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more,99% or more, or 100% sequence identity) with a C2c10 or C2c9protein. Insome cases a type V CRISPR/Cas effector protein comprises an amino acidsequence having 90% or more sequence identity (e.g., 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with a C2c10or C2c9protein. In some cases a type V CRISPR/Cas effector proteincomprises a C2c10 or C2c9amino acid sequence.

In some cases, a subject type V CRISPR/Cas effector protein (e.g., aCas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) is fusedto (conjugated to) a heterologous polypeptide. In some cases, aheterologous polypeptide (a fusion partner) provides for subcellularlocalization, i.e., the heterologous polypeptide contains a subcellularlocalization sequence (e.g., a nuclear localization signal (NLS) fortargeting to the nucleus, a sequence to keep the fusion protein out ofthe nucleus, e.g., a nuclear export sequence (NES), a sequence to keepthe fusion protein retained in the cytoplasm, a mitochondriallocalization signal for targeting to the mitochondria, a chloroplastlocalization signal for targeting to a chloroplast, an ER retentionsignal, and the like). In some cases, a type V CRISPR/Cas effectorprotein (e.g., a Cas12 protein) does not include a NLS so that theprotein is not targeted to the nucleus (which can be advantageous, e.g.,when it desirable to cleave non-target ssDNAs in the cytosol). In somecases, the heterologous polypeptide can provide a tag (i.e., theheterologous polypeptide is a detectable label) for ease of trackingand/or purification (e.g., a fluorescent protein, e.g., greenfluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and thelike; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; aFLAG tag; a Myc tag; and the like).

In some cases a type V CRISPR/Cas effector protein (e.g., a Cas12protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) includes (isfused to) a nuclear localization signal (NLS) (e.g., in some cases 2 ormore, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some cases, atype V CRISPR/Cas effector protein includes one or more NLSs (e.g., 2 ormore, 3 or more, 4 or more, or 5 or more NLSs). In some cases, one ormore NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) arepositioned at or near (e.g., within 50 amino acids of) the N-terminusand/or the C-terminus. In some cases, one or more NLSs (2 or more, 3 ormore, 4 or more, or 5 or more NLSs) are positioned at or near (e.g.,within 50 amino acids of) the N-terminus. In some cases, one or moreNLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positionedat or near (e.g., within 50 amino acids of) the C-terminus. In somecases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) arepositioned at or near (e.g., within 50 amino acids of) both theN-terminus and the C-terminus. In some cases, an NLS is positioned atthe N-terminus and an NLS is positioned at the C-terminus.

In some cases a type V CRISPR/Cas effector protein (e.g., a Cas12protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) includes (isfused to) between 1 and 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10,2-9, 2-8, 2-7, 2-6, or 2-5 NLSs). In some cases a type V CRISPR/Caseffector protein includes (is fused to) between 2 and 5 NLSs (e.g., 2-4,or 2-3 NLSs). Non-limiting examples of NLSs include an NLS sequencederived from: the NLS of the SV40 virus large T-antigen, having theamino acid sequence PKKKRKV (SEQ ID NO: 136); the NLS from nucleoplasmin(e.g., the nucleoplasmin bipartite NLS with the sequenceKRPAATKKAGQAKKKK (SEQ ID NO: 137)); the c-myc NLS having the amino acidsequence PAAKRVKLD (SEQ ID NO: 138) or RQRRNELKRSP (SEQ ID NO: 139); thehRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 140); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 141) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 142) andPPKKARED (SEQ ID NO: 143) of the myoma T protein; the sequence PQPKKKPL(SEQ ID NO: 144) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO:145) of mouse c-ab1 IV; the sequences DRLRR (SEQ ID NO: 146) and PKQKKRK(SEQ ID NO: 147) of the influenza virus NS1; the sequence RKLKKKIKKL(SEQ ID NO: 148) of the Hepatitis virus delta antigen; the sequenceREKKKFLKRR (SEQ ID NO: 149) of the mouse Mxl protein; the sequenceKRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 150) of the human poly(ADP-ribose)polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 151) of thesteroid hormone receptors (human) glucocorticoid. In general, NLS (ormultiple NLSs) are of sufficient strength to drive accumulation of theprotein in a detectable amount in the nucleus of a eukaryotic cell.Detection of accumulation in the nucleus may be performed by anysuitable technique. Protospacer Adjacent Motif (PAM)

A Type V CRISPR/Cas effector protein binds to target DNA at a targetsequence defined by the region of complementarity between theDNA-targeting RNA and the target DNA. As is the case for many CRISPR/Casendonucleases, site-specific binding (and/or cleavage) of a doublestranded target DNA occurs at locations determined by both (i)base-pairing complementarity between the guide RNA and the target DNA;and (ii) a short motif [referred to as the protospacer adjacent motif(PAM)] in the target DNA.

In some cases, the PAM for a Type V CRISPR/Cas effector protein isimmediately 5′ of the target sequence (e.g., of the non-complementarystrand of the target DNA—the complementary strand hybridizes to theguide sequence of the guide RNA while the non-complementary strand doesnot directly hybridize with the guide RNA and is the reverse complementof the non-complementary strand). In some cases (e.g., when Casl2a orCasl2b as described herein is used), the PAM sequence is 5′-TTN-3′. Insome cases, the PAM sequence is 5′-TTTN-3.' (e.g., see FIG. 2).

In some cases, different Type V CRISPR/Cas effector proteins (i.e., TypeV CRISPR/Cas effector proteins from various species) may be advantageousto use in the various provided methods in order to capitalize on adesired feature (e.g., specific enzymatic characteristics of differentType V CRISPR/Cas effector proteins). Type V CRISPR/Cas effectorproteins from different species may require different PAM sequences inthe target DNA. Thus, for a particular Type V CRISPR/Cas effectorprotein of choice, the PAM sequence requirement may be different thanthe 5′-TTN-3′ or 5′-TTTN-3′ sequence described above. Various methods(including in silico and/or wet lab methods) for identification of theappropriate PAM sequence are known in the art and are routine, and anyconvenient method can be used.

Guide RNA

A nucleic acid molecule (e.g., a natural crRNA) that binds to a type VCRISPR/Cas effector protein (e.g., a Casl2 protein such as Casl2a,Casl2b, Casl2c, Casl2d, Casl2e), forming a ribonucleoprotein complex(RNP), and targets the complex to a specific target sequence within atarget DNA is referred to herein as a “guide RNA.” It is to beunderstood that in some cases, a hybrid DNA/RNA can be made such that aguide RNA includes DNA bases in addition to RNA bases—but the term“guide RNA” is still used herein to encompass such hybrid molecules. Asubject guide RNA includes a guide sequence (also referred to as a“spacer”)(that hybridizes to target sequence of a target DNA) and aconstant region (e.g., a region that is adjacent to the guide sequenceand binds to the type V CRISPR/Cas effector protein). A “constantregion” can also be referred to herein as a “protein-binding segment.”In some cases, e.g., for Casl2a, the constant region is 5′ of the guidesequence.

Guide Sequence

The guide sequence has complementarity with (hybridizes to) a targetsequence of the target DNA. In some cases, the guide sequence is 15-28nucleotides (nt) in length (e.g., 15-26, 15-24, 15-22, 15-20, 15-18,16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20,17-18, 18-26, 18-24, or 18-22 nt in length). In some cases, the guidesequence is 18-24 nucleotides (nt) in length. In some cases, the guidesequence is at least 15 nt long (e.g., at least 16, 18, 20, or 22 ntlong). In some cases, the guide sequence is at least 17 nt long. In somecases, the guide sequence is at least 18 nt long. In some cases, theguide sequence is at least 20 nt long.

In some cases, the guide sequence has 80% or more (e.g., 85% or more,90% or more, 95% or more, or 100% complementarity) with the targetsequence of the target DNA. In some cases, the guide sequence is 100%complementary to the target sequence of the target DNA. In some cases,the target DNA includes at least 15 nucleotides (nt) of complementaritywith the guide sequence of the guide RNA.

Constant Region

Examples of constant regions for guide RNAs that can be used with a typeV CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a,Cas12b, Cas12c, Cas12d, Cas12e) are presented in FIG. 2.

In some cases, a subject guide RNA includes a nucleotide sequence having70% or more identity (e.g., 80% or more, 85% or more, 90% or more, 95%or more, 98% or more, 99% or more, or 100% identity) with any one of thecrRNA repeat sequences set forth in FIG. 2. In some cases, a subjectguide RNA includes a nucleotide sequence having 90% or more identity(e.g., 95% or more, 98% or more, 99% or more, or 100% identity) with anyone of the crRNA repeat sequences set forth in FIG. 2. In some cases, asubject guide RNA includes a crRNA nucleotide sequence set forth in FIG.2.

In some cases, the guide RNA includes a double stranded RNA duplex(dsRNA duplex). In some cases, a guide RNA includes a dsRNA duplex witha length of from 2 to 12 bp (e.g., from 2 to 10 bp, 2 to 8 bp, 2 to 6bp, 2 to 5 bp, 2 to 4 bp, 3 to 12 bp, 3 to 10 bp, 3 to 8 bp, 3 to 6 bp,3 to 5 bp, 3 to 4 bp, 4 to 12 bp, 4 to 10 bp, 4 to 8 bp, 4 to 6 bp, or 4to 5 bp). In some cases, a guide RNA includes a dsRNA duplex that is 2or more bp in length (e.g., 3 or more, 4 or more, 5 or more, 6 or more,or 7 or more bp in length). In some cases, a guide RNA includes a dsRNAduplex that is longer than the dsRNA duplex of a corresponding wild typeguide RNA. In some cases, a guide RNA includes a dsRNA duplex that isshorter than the dsRNA duplex of a corresponding wild type guide RNA.

In some cases, the constant region of a guide RNA is 15 or morenucleotides (nt) in length (e.g., 18 or more, 20 or more, 21 or more, 22or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28or more, 29 or more, 30 or more, 31 or more nt, 32 or more, 33 or more,34 or more, or 35 or more nt in length). In some cases, the constantregion of a guide RNA is 18 or more nt in length.

In some cases, the constant region of a guide RNA has a length in arange of from 12 to 100 nt (e.g., from 12 to 90, 12 to 80, 12 to 70, 12to 60, 12 to 50, 12 to 40, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15to 60, 15 to 50, 15 to 40, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20to 60, 20 to 50, 20 to 40, 25 to 100, 25 to 90, 25 to 80, 25 to 70, 25to 60, 25 to 50, 25 to 40, 28 to 100, 28 to 90, 28 to 80, 28 to 70, 28to 60, 28 to 50, 28 to 40, 29 to 100, 29 to 90, 29 to 80, 29 to 70, 29to 60, 29 to 50, or 29 to 40 nt). In some cases, the constant region ofa guide RNA has a length in a range of from 28 to 100 nt. In some cases,the region of a guide RNA that is 5′ of the guide sequence has a lengthin a range of from 28 to 40 nt.

In some cases, the constant region of a guide RNA is truncated relativeto (shorter than) the corresponding region of a corresponding wild typeguide RNA. In some cases, the constant region of a guide RNA is extendedrelative to (longer than) the corresponding region of a correspondingwild type guide RNA. In some cases, a subject guide RNA is 30 or morenucleotides (nt) in length (e.g., 34 or more, 40 or more, 45 or more, 50or more, 55 or more, 60 or more, 65 or more, 70 or more, or 80 or morent in length). In some cases, the guide RNA is 35 or more nt in length.

Precursor Guide RNA Array

A Type V CRISPR/Cas effector protein (e.g., a Cas12 protein such asCas12a, Cas12b, Cas12c, Cas12d, Cas12e) can cleave a precursor guide RNAinto a mature guide RNA, e.g., by endoribonucleolytic cleavage of theprecursor. A Type V CRISPR/Cas effector protein (e.g., a Cas12 proteinsuch as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) can cleave a precursorguide RNA array (that includes more than one guide RNA arrayed intandem) into two or more individual guide RNAs. Thus, in some cases aprecursor guide RNA array comprises two or more (e.g., 3 or more, 4 ormore, 5 or more, 2, 3, 4, or 5) guide RNAs (e.g., arrayed in tandem asprecursor molecules). In other words, in some cases, two or more guideRNAs can be present on an array (a precursor guide RNA array). A Type VCRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a,Cas12b, Cas12c, Cas12d, Cas12e) can cleave the precursor guide RNA arrayinto individual guide RNAs

In some cases a subject guide RNA array includes 2 or more guide RNAs(e.g., 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, guideRNAs). The guide RNAs of a given array can target (i.e., can includeguide sequences that hybridize to) different target sites of the sametarget DNA (e.g., which can increase sensitivity of detection) and/orcan target different target DNA molecules (e.g., single nucleotidepolymorphisms (SNPs), different strains of a particular virus, etc.),and such could be used for example to detect multiple strains of avirus. In some cases, each guide RNA of a precursor guide RNA array hasa different guide sequence. In some cases, two or more guide RNAs of aprecursor guide RNA array have the same guide sequence.

In some cases, the precursor guide RNA array comprises two or more guideRNAs that target different target sites within the same target DNAmolecule. For example, such a scenario can in some cases increasesensitivity of detection by activating Type V CRISPR/Cas effectorprotein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d,Cas12e) when either one hybridizes to the target DNA molecule. As such,in some cases as subject composition (e.g., kit) or method includes twoor more guide RNAs (in the context of a precursor guide RNA array, ornot in the context of a precursor guide RNA array, e.g., the guide RNAscan be mature guide RNAs).

In some cases, the precursor guide RNA array comprises two or more guideRNAs that target different target DNA molecules. For example, such ascenario can result in a positive signal when any one of a family ofpotential target DNAs is present. Such an array could be used fortargeting a family of transcripts, e.g., based on variation such assingle nucleotide polymorphisms (SNPs) (e.g., for diagnostic purposes).Such could also be useful for detecting whether any one of a number ofdifferent strains of virus is present. Such could also be useful fordetecting whether any one of a number of different species, strains,isolates, or variants of a bacterium is present (e.g., differentspecies, strains, isolates, or variants of Mycobacterium, differentspecies, strains, isolates, or variants of Neisseria, different species,strains, isolates, or variants of Staphylococcus aureus; differentspecies, strains, isolates, or variants of E. coli; etc.). As such, insome cases as subject composition (e.g., kit) or method includes two ormore guide RNAs (in the context of a precursor guide RNA array, or notin the context of a precursor guide RNA array, e.g., the guide RNAs canbe mature guide RNAs).

Nucleic Acid Modifications

In some cases, a labeled detector ssDNA (and/or a guide RNA) comprisesone or more modifications, e.g., a base modification, a backbonemodification, a sugar modification, etc., to provide the nucleic acidwith a new or enhanced feature (e.g., improved stability). As is knownin the art, a nucleoside is a base-sugar combination. The base portionof the nucleoside is normally a heterocyclic base. The two most commonclasses of such heterocyclic bases are the purines and the pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, the 3′, or the 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turn,the respective ends of this linear polymeric compound can be furtherjoined to form a circular compound, however, linear compounds aregenerally suitable. In addition, linear compounds may have internalnucleotide base complementarity and may therefore fold in a manner as toproduce a fully or partially double-stranded compound. Withinoligonucleotides, the phosphate groups are commonly referred to asforming the internucleoside backbone of the oligonucleotide. The normallinkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Modified Backbones and Modified Internucleoside Linkages

Examples of suitable modifications include modified nucleic acidbackbones and non-natural internucleoside linkages. Nucleic acids havingmodified backbones include those that retain a phosphorus atom in thebackbone and those that do not have a phosphorus atom in the backbone.

Suitable modified oligonucleotide backbones containing a phosphorus atomtherein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be a basic (the nucleobase is missing or has ahydroxyl group in place thereof). Various salts (such as, for example,potassium or sodium), mixed salts and free acid forms are also included.

In some cases, a labeled detector ssDNA (and/or a guide RNA) comprisesone or more phosphorothioate and/or heteroatom internucleoside linkages,in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—(known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—(wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). MMI type internucleoside linkages are disclosed inthe above referenced U.S. Pat. No. 5,489,677. Suitable amideinternucleoside linkages are disclosed in t U.S. Pat. No. 5,602,240.

Also suitable are nucleic acids having morpholino backbone structures asdescribed in, e.g., U.S. Pat. No. 5,034,506. For example, in some cases,a labeled detector ssDNA (and/or a guide RNA) comprises a 6-memberedmorpholino ring in place of a ribose ring. In some cases, aphosphorodiamidate or other non-phosphodiester internucleoside linkagereplaces a phosphodiester linkage.

Suitable modified polynucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Mimetics

A labeled detector ssDNA (and/or a guide RNA) can be a nucleic acidmimetic. The term “mimetic” as it is applied to polynucleotides isintended to include polynucleotides wherein only the furanose ring orboth the furanose ring and the internucleotide linkage are replaced withnon-furanose groups, replacement of only the furanose ring is alsoreferred to in the art as being a sugar surrogate. The heterocyclic basemoiety or a modified heterocyclic base moiety is maintained forhybridization with an appropriate target nucleic acid. One such nucleicacid, a polynucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA, the sugar-backbone of a polynucleotide is replaced withan amide containing backbone, in particular an aminoethylglycinebackbone. The nucleotides are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.

One polynucleotide mimetic that has been reported to have excellenthybridization properties is a peptide nucleic acid (PNA). The backbonein PNA compounds is two or more linked aminoethylglycine units whichgives PNA an amide containing backbone. The heterocyclic base moietiesare bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative U.S. patents that describe thepreparation of PNA compounds include, but are not limited to: U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262.

Another class of polynucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. One class of linking groups has been selected to give anon-ionic oligomeric compound. The non-ionic morpholino-based oligomericcompounds are less likely to have undesired interactions with cellularproteins. Morpholino-based polynucleotides are non-ionic mimics ofoligonucleotides which are less likely to form undesired interactionswith cellular proteins (Dwaine A. Braasch and David R. Corey,Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotidesare disclosed in U.S. Pat. No. 5,034,506. A variety of compounds withinthe morpholino class of polynucleotides have been prepared, having avariety of different linking groups joining the monomeric subunits.

A further class of polynucleotide mimetic is referred to as cyclohexenylnucleic acids (CeNA). The furanose ring normally present in a DNA/RNAmolecule is replaced with a cyclohexenyl ring. CeNA DMT protectedphosphoramidite monomers have been prepared and used for oligomericcompound synthesis following classical phosphoramidite chemistry. Fullymodified CeNA oligomeric compounds and oligonucleotides having specificpositions modified with CeNA have been prepared and studied (see Wang etal., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general theincorporation of CeNA monomers into a DNA chain increases its stabilityof a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA andDNA complements with similar stability to the native complexes. Thestudy of incorporating CeNA structures into natural nucleic acidstructures was shown by NMR and circular dichroism to proceed with easyconformational adaptation.

A further modification includes Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ringthereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming abicyclic sugar moiety. The linkage can be a methylene (—CH₂—), groupbridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2(Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogsdisplay very high duplex thermal stabilities with complementary DNA andRNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradationand good solubility properties. Potent and nontoxic antisenseoligonucleotides containing LNAs have been described (Wahlestedt et al.,Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

Modified Sugar Moieties

A labeled detector ssDNA (and/or a guide RNA) can also include one ormore substituted sugar moieties. Suitable polynucleotides comprise asugar substituent group selected from: OH; F; O—, S—, or N-alkyl; O—,S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly suitable areO((CH₂)_(n)O)_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n), CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from1 to about 10. Other suitable polynucleotides comprise a sugarsubstituent group selected from: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A suitable modification includes 2′-methoxyethoxy (2′-O—CH₂ CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Hely.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furthersuitable modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other suitable sugar substituent groups include methoxy (—O—CH₃),aminopropoxy (——O CH₂ CH₂ CH₂NH₂), allyl (—CH₂—CH═CH₂), —O—allyl(——O——CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be inthe arabino (up) position or ribo (down) position. A suitable 2′-arabinomodification is 2′-F. Similar modifications may also be made at otherpositions on the oligomeric compound, particularly the 3′ position ofthe sugar on the 3′ terminal nucleoside or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligomeric compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar.

Base Modifications and Substitutions

A labeled detector ssDNA (and/or a guide RNA) may also includenucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are useful for increasing the binding affinity of anoligomeric compound. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi et al., eds., AntisenseResearch and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) andare suitable base substitutions, e.g., when combined with2′-O-methoxyethyl sugar modifications.

Introducing Components Into a Target Cell

A guide RNA (or a nucleic acid comprising a nucleotide sequence encodingsame) and/or a type V CRISPR/Cas effector protein can be introduced intoa host cell by any of a variety of well-known methods. As a non-limitingexample, a guide RNA and/or type V CRISPR/Cas effector protein can becombined with a lipid. As another non-limiting example, a guide RNAand/or type V CRISPR/Cas effector protein can be combined with aparticle, or formulated into a particle.

Methods of introducing a nucleic acid and/or protein into a host cellare known in the art, and any convenient method can be used to introducea subject nucleic acid (e.g., an expression construct/vector) into atarget cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animalcell, mammalian cell, human cell, and the like). Suitable methodsinclude, e.g., viral infection, transfection, conjugation, protoplastfusion, lipofection, electroporation, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro injection,nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al.Adv Drug Deliv Rev. 2012 Sep. 13. pii: S0169-409X(12)00283-9. doi:10.1016/j.addr.2012.09.023), and the like.

A guide RNA can be introduced, e.g., as a DNA molecule encoding theguide RNA, or can be provided directly as an RNA molecule (or a hybridmolecule when applicable). In some cases, a type V CRISPR/Cas effectorprotein is provided as a nucleic acid (e.g., an mRNA, a DNA, a plasmid,an expression vector, a viral vector, etc.) that encodes the protein. Insome cases, the type V CRISPR/Cas effector protein is provided directlyas a protein (e.g., without an associated guide RNA or with an associateguide RNA, i.e., as a ribonucleoprotein complex-RNP). Like a guide RNA,a type V CRISPR/Cas effector protein can be introduced into a cell(provided to the cell) by any convenient method; such methods are knownto those of ordinary skill in the art. As an illustrative example, atype V CRISPR/Cas effector protein can be injected directly into a cell(e.g., with or without a guide RNA or nucleic acid encoding a guideRNA). As another example, a preformed complex of a type V CRISPR/Caseffector protein and a guide RNA (an RNP) can be introduced into a cell(e.g., eukaryotic cell) (e.g., via injection, via nucleofection; via aprotein transduction domain (PTD) conjugated to one or more components,e.g., conjugated to the type V CRISPR/Cas effector protein, conjugatedto a guide RNA; etc.).

In some cases, a nucleic acid (e.g., a guide RNA; a nucleic acidcomprising a nucleotide sequence encoding a type V CRISPR/Cas effectorprotein; etc.) and/or a polypeptide (e.g., a type V CRISPR/Cas effectorprotein) is delivered to a cell (e.g., a target host cell) in aparticle, or associated with a particle. The terms “particle” and“nanoparticle” can be used interchangeably, as appropriate.

This can be achieved, e.g., using particles or lipid envelopes, e.g., aribonucleoprotein (RNP) complex can be delivered via a particle, e.g., adelivery particle comprising lipid or lipidoid and hydrophilic polymer,e.g., a cationic lipid and a hydrophilic polymer, for instance whereinthe cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP) or 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/orwherein the hydrophilic polymer comprises ethylene glycol orpolyethylene glycol (PEG); and/or wherein the particle further comprisescholesterol (e.g., particle from formulation 1=DOTAP 100, DMPC 0, PEG 0,Cholesterol 0; formulation number 2=DOTAP 90, DMPC 0, PEG 10,Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol5).

A type V CRISPR/Cas effector protein (or an mRNA comprising a nucleotidesequence encoding the protein) and/or guide RNA (or a nucleic acid suchas one or more expression vectors encoding the guide RNA) may bedelivered simultaneously using particles or lipid envelopes. Forexample, a biodegradable core-shell structured nanoparticle with a poly(β-amino ester) (PBAE) core enveloped by a phospholipid bilayer shellcan be used. In some cases, particles/nanoparticles based on selfassembling bioadhesive polymers are used; such particles/nanoparticlesmay be applied to oral delivery of peptides, intravenous delivery ofpeptides and nasal delivery of peptides, e.g., to the brain. Otherembodiments, such as oral absorption and ocular delivery of hydrophobicdrugs are also contemplated. A molecular envelope technology, whichinvolves an engineered polymer envelope which is protected and deliveredto the site of the disease, can be used. Doses of about 5 mg/kg can beused, with single or multiple doses, depending on various factors, e.g.,the target tissue.

Lipidoid compounds (e.g., as described in US patent publication20110293703) are also useful in the administration of polynucleotides,and can be used. In one aspect, aminoalcohol lipidoid compounds arecombined with an agent to be delivered to a cell or a subject to formmicroparticles, nanoparticles, liposomes, or micelles. The aminoalcohollipidoid compounds may be combined with other aminoalcohol lipidoidcompounds, polymers (synthetic or natural), surfactants, cholesterol,carbohydrates, proteins, lipids, etc. to form the particles. Theseparticles may then optionally be combined with a pharmaceuticalexcipient to form a pharmaceutical composition.

A poly(beta-amino alcohol) (PBAA) can be used, sugar-based particles maybe used, for example GalNAc, as described with reference to WO2014118272(incorporated herein by reference) and Nair, J K et al., 2014, Journalof the American Chemical Society 136 (49), 16958-16961). In some cases,lipid nanoparticles (LNPs) are used. Spherical Nucleic Acid (SNA™)constructs and other nanoparticles (particularly gold nanoparticles) canbe used to a target cell. See, e.g., Cutler et al., J. Am. Chem. Soc.2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al.,ACS Nano. 2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et al.,Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin, Nanomedicine 20127:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691,Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci.USA. 2013 110(19): 7625-7630, Jensen et al., Sci. Transl. Med. 5,209ra152 (2013) and Mirkin, et al., Small, 10:186-192. Semi-solid andsoft nanoparticles are also suitable for delivery. An exosome can beused for delivery. Exosomes are endogenous nano-vesicles that transportRNAs and proteins, and which can deliver RNA to the brain and othertarget organs. Supercharged proteins can be used for delivery to a cell.Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoreticalcharge. Both supernegatively and superpositively charged proteinsexhibit the ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can facilitate the functional delivery ofthese macromolecules into mammalian cells both in vitro and in vivo.Cell Penetrating Peptides (CPPs) can be used for delivery. CPPstypically have an amino acid composition that either contains a highrelative abundance of positively charged amino acids such as lysine orarginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids.

Target Cells of Interest

Suitable target cells (which can comprise target nucleic acids such asgenomic DNA) include, but are not limited to: a bacterial cell; anarchaeal cell; a cell of a single-cell eukaryotic organism; a plantcell; an algal cell, e.g., Botryococcus braunii, Chlamydomonasreinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassumpatens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); ananimal cell; a cell from an invertebrate animal (e.g. fruit fly, acnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g.,a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid(e.g., a spider; a tick; etc.); a cell from a vertebrate animal (e.g., afish, an amphibian, a reptile, a bird, a mammal); a cell from a mammal(e.g., a cell from a rodent; a cell from a human; a cell of a non-humanmammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph(e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel,a llama, a vicuña, a sheep, a goat, etc.); a cell of a marine mammal(e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.)and the like. Any type of cell may be of interest (e.g. a stem cell,e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS)cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, aspermatogonia, etc.), an adult stem cell, a somatic cell, e.g. afibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell,a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cellof an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc.stage zebrafish embryo; etc.).

Cells may be from established cell lines or they may be primary cells,where “primary cells”, “primary cell lines”, and “primary cultures” areused interchangeably herein to refer to cells and cells cultures thathave been derived from a subject and allowed to grow in vitro for alimited number of passages, i.e. splittings, of the culture. Forexample, primary cultures are cultures that may have been passaged 0times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but notenough times go through the crisis stage. Typically, the primary celllines are maintained for fewer than 10 passages in vitro. Target cellscan be unicellular organisms and/or can be grown in culture. If thecells are primary cells, they may be harvest from an individual by anyconvenient method. For example, leukocytes may be conveniently harvestedby apheresis, leukocytapheresis, density gradient separation, etc.,while cells from tissues such as skin, muscle, bone marrow, spleen,liver, pancreas, lung, intestine, stomach, etc. can be convenientlyharvested by biopsy.

Because the guide RNA provides specificity by hybridizing to targetnucleic acid, a mitotic and/or post-mitotic cell of interest in thedisclosed methods may include a cell of any organism (e.g. a bacterialcell, an archaeal cell, a cell of a single-cell eukaryotic organism, aplant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonasreinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassumpatens, C. agardh, and the like, a fungal cell (e.g., a yeast cell), ananimal cell, a cell of an invertebrate animal (e.g. fruit fly,cnidarian, echinoderm, nematode, etc.), a cell of a vertebrate animal(e.g., fish, amphibian, reptile, bird, mammal), a cell of a mammal, acell of a rodent, a cell of a human, etc.).

Plant cells include cells of a monocotyledon, and cells of adicotyledon. The cells can be root cells, leaf cells, cells of thexylem, cells of the phloem, cells of the cambium, apical meristem cells,parenchyma cells, collenchyma cells, sclerenchyma cells, and the like.Plant cells include cells of agricultural crops such as wheat, corn,rice, sorghum, millet, soybean, etc. Plant cells include cells ofagricultural fruit and nut plants, e.g., plant that produce apricots,oranges, lemons, apples, plums, pears, almonds, etc.

Non-limiting examples of cells (target cells) include: a prokaryoticcell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of asingle-cell eukaryotic organism, a protozoa cell, a cell from a plant(e.g., cells from plant crops, fruits, vegetables, grains, soy bean,corn, maize, wheat, seeds, tomatos, rice, cassava, sugarcane, pumpkin,hay, potatos, cotton, cannabis, tobacco, flowering plants, conifers,gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts,mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g.,Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsisgaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and thelike), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cellfrom a mushroom), an animal cell, a cell from an invertebrate animal(e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from avertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cellfrom a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep);a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline(e.g., a cat); a canine (e.g., a dog); etc.), and the like. In somecases, the cell is a cell that does not originate from a naturalorganism (e.g., the cell can be a synthetically made cell; also referredto as an artificial cell).

A cell can be an in vitro cell (e.g., established cultured cell line). Acell can be an ex vivo cell (cultured cell from an individual). A cellcan be and in vivo cell (e.g., a cell in an individual). A cell can bean isolated cell. A cell can be a cell inside of an organism. A cell canbe an organism.

Suitable cells include human embryonic stem cells, fetal cardiomyocytes,myofibroblasts, mesenchymal stem cells, autotransplated expandedcardiomyocytes, adipocytes, totipotent cells, pluripotent cells, bloodstem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymalcells, embryonic stem cells, parenchymal cells, epithelial cells,endothelial cells, mesothelial cells, fibroblasts, osteoblasts,chondrocytes, exogenous cells, endogenous cells, stem cells,hematopoietic stem cells, bone-marrow derived progenitor cells,myocardial cells, skeletal cells, fetal cells, undifferentiated cells,multi-potent progenitor cells, unipotent progenitor cells, monocytes,cardiac myoblasts, skeletal myoblasts, macrophages, capillaryendothelial cells, xenogenic cells, allogenic cells, and post-natal stemcells.

In some cases, the cell is an immune cell, a neuron, an epithelial cell,and endothelial cell, or a stem cell. In some cases, the immune cell isa T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell,or a macrophage. In some cases, the immune cell is a cytotoxic T cell.In some cases, the immune cell is a helper T cell. In some cases, theimmune cell is a regulatory T cell (Treg).

In some cases, the cell is a stem cell. Stem cells include adult stemcells. Adult stem cells are also referred to as somatic stem cells.

Adult stem cells are resident in differentiated tissue, but retain theproperties of self-renewal and ability to give rise to multiple celltypes, usually cell types typical of the tissue in which the stem cellsare found. Numerous examples of somatic stem cells are known to those ofskill in the art, including muscle stem cells; hematopoietic stem cells;epithelial stem cells; neural stem cells; mesenchymal stem cells;mammary stem cells; intestinal stem cells; mesodermal stem cells;endothelial stem cells; olfactory stem cells; neural crest stem cells;and the like.

Stem cells of interest include mammalian stem cells, where the term“mammalian” refers to any animal classified as a mammal, includinghumans; non-human primates; domestic and farm animals; and zoo,laboratory, sports, or pet animals, such as dogs, horses, cats, cows,mice, rats, rabbits, etc. In some cases, the stem cell is a human stemcell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat)stem cell. In some cases, the stem cell is a non-human primate stemcell.

Kits

The present disclosure provides a kit for detecting a target DNA, e.g.,in a sample comprising a plurality of DNAs. In some cases, the kitcomprises: (a) a labeled detector ssDNA (e.g., a labeled detector ssDNAcomprising a fluorescence-emitting dye pair, i.e., a FRET pair and/or aquencher/fluor pair); and (b) one or more of: (i) a guide RNA, and/or anucleic acid encoding said guide RNA; (ii); a precursor guide RNA arraycomprising two or more guide RNAs (e.g., each of which has a differentguide sequence), and/or a nucleic acid encoding the precursor guide RNAarray; and (iii) a Type V CRISPR/Cas effector protein (e.g., a Cas12protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e), and/or anucleic acid encoding said Type V CRISPR/Cas effector protein. In somecases a nucleic acid encoding a precursor guide RNA array includessequence insertion sites for the insertion of guide sequences by a user.

In some cases, a subject kit comprises: (a) a labeled detector ssDNAcomprising a fluorescence-emitting dye pair, i.e., a FRET pair and/or aquencher/fluor pair; and (b) one or more of: (i) a guide RNA, and/or anucleic acid encoding said guide RNA; (ii); a precursor guide RNA arraycomprising two or more guide RNAs (e.g., each of which has a differentguide sequence), and/or a nucleic acid encoding the precursor guide RNAarray; and (iii) a Type V CRISPR/Cas effector protein (e.g., a Cas12protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e), and/or anucleic acid encoding said Type V CRISPR/Cas effector protein.

Positive Controls

A kit of the present disclosure (e.g., one that comprises a labeleddetector ssDNA and a type V CRISPR/Cas effector protein) can alsoinclude a positive control target DNA. In some cases, the kit alsoincludes a positive control guide RNA that comprises a nucleotidesequence that hybridizes to the control target DNA. In some cases, thepositive control target DNA is provided in various amounts, in separatecontainers. In some cases, the positive control target DNA is providedin various known concentrations, in separate containers, along withcontrol non-target DNAs.

Nucleic Acids

While the RNAs of the disclosure (e.g., guide RNAs and precursor guideRNA arrays) can be synthesized using any convenient method (e.g.,chemical synthesis, in vitro using an RNA polymerase enzyme, e.g., T7polymerase, T3 polymerase, SP6 polymerase, etc.), nucleic acids encodingguide RNAs and/or precursor guide RNA arrays are also envisioned.Additionally, while Type V CRISPR/Cas effector proteins (e.g., a Cas12protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) of thedisclosure can be provided (e.g., as part of a kit) in protein form,nucleic acids (such as mRNA and/or DNA) encoding the Type V CRISPR/Caseffector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c,Cas12d, Cas12e)(s) can also be provided.

For example, in some cases, a kit of the present disclosure comprises anucleic acid (e.g., a DNA, e.g., a recombinant expression vector) thatcomprises a nucleotide sequence encoding a guide RNA. In some cases, thenucleotide sequence encodes a guide RNA without a guide sequence. Forexample, in some cases, the nucleic acid comprises a nucleotide sequenceencoding a constant region of a guide RNA (a guide RNA without a guidesequence), and comprises an insertion site for a nucleic acid encoding aguide sequence. In some cases, a kit of the present disclosure comprisesa nucleic acid (e.g., an mRNA, a DNA, e.g., a recombinant expressionvector) that comprises a nucleotide sequence encoding a Type VCRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a,Cas12b, Cas12c, Cas12d, Cas12e).

In some cases, a kit of the present disclosure comprises a nucleic acid(e.g., a DNA, e.g., a recombinant expression vector) that comprises anucleotide sequence encoding a precursor guide RNA array (e.g., in somecases where each guide RNA of the array has a different guide sequence).In some cases, one or more of the encoded guide RNAs of the array doesnot have a guide sequence, e.g., the nucleic acid can include insertionsite(s) for the guide sequence(s) of one or more of the guide RNAs ofthe array. In some cases, a subject guide RNA can include a handle froma precursor crRNA but does not necessarily have to include multipleguide sequences.

In some cases, the guide RNA-encoding nucleotide sequence (and/or theprecursor guide RNA array-encoding nucleotide sequence) is operablylinked to a promoter, e.g., a promoter that is functional in aprokaryotic cell, a promoter that is functional in a eukaryotic cell, apromoter that is functional in a mammalian cell, a promoter that isfunctional in a human cell, and the like. In some cases, a nucleotidesequence encoding a Type V CRISPR/Cas effector protein (e.g., a Cas12protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) is operablylinked to a promoter, e.g., a promoter that is functional in aprokaryotic cell, a promoter that is functional in a eukaryotic cell, apromoter that is functional in a mammalian cell, a promoter that isfunctional in a human cell, a cell type-specific promoter, a regulatablepromoter, a tissue-specific promoter, and the like.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-45 (SET A) and1-54 (SET B) are provided below. As will be apparent to those of skillin the art upon reading this disclosure, each of the individuallynumbered aspects may be used or combined with any of the preceding orfollowing individually numbered aspects. This is intended to providesupport for all such combinations of aspects and is not limited tocombinations of aspects explicitly provided below:

Set A

1. A method of detecting a target DNA in a sample, the methodcomprising:

-   -   (a) contacting the sample with:        -   (i) a type V CRISPR/Cas effector protein;        -   (ii) a guide RNA comprising: a region that binds to the type            V CRISPR/Cas effector protein, and a guide sequence that            hybridizes with the target DNA; and            -   (iii) a detector DNA that is single stranded and does                not hybridize with the guide sequence of the guide RNA;                and    -   (b) measuring a detectable signal produced by cleavage of the        single stranded detector DNA by the type V CRISPR/Cas effector        protein, thereby detecting the target DNA.

2. The method of 1, comprising contacting the sample with a precursorguide RNA array, wherein the type V CRISPR/Cas effector protein cleavesthe precursor guide RNA array to produce said guide RNA and at least oneadditional guide RNA.

3. The method of 1 or 2, wherein the target DNA is single stranded.

4. The method of 1 or 2, wherein the target DNA is double stranded.

5. The method of any one of 1-4, wherein the target DNA is viral DNA.

6. The method of any one of 1-4, wherein the target DNA is papovavirus,hepdnavirus, herpesvirus, adenovirus, poxvirus, or parvovirus DNA.

7. The method of any one of 1-4, wherein the type V CRISPR/Cas effectorprotein is a Cas12 protein.

8. The method of any one of 1-6, wherein the type V CRISPR/Cas effectorprotein is a Cas12a (Cpf1) or Cas12b (C2c1) protein.

9. The method according to any one of 1-8, wherein the sample comprisesDNA molecules from a cell lysate.

10. The method according to any one of 1-9, wherein the sample comprisescells.

11. The method according to any one of 1-10, wherein said contacting iscarried out inside of a cell in vitro, ex vivo, or in vivo.

12. The method according to 11, wherein the cell is a eukaryotic cell.

13. The method according to any one of 1-12, wherein the target DNA canbe detected at a concentration as low as 200 fM.

14. The method according to any one of 1-13, comprising determining anamount of the target DNA present in the sample.

15. The method according to 14, wherein said determining comprises:

-   -   measuring the detectable signal to generate a test measurement;    -   measuring a detectable signal produced by a reference sample or        cell to generate a reference measurement; and    -   comparing the test measurement to the reference measurement to        determine an amount of target DNA present in the sample.

16. The method according to any one of 1-15, wherein measuring adetectable signal comprises one or more of: gold nanoparticle baseddetection, fluorescence polarization, colloid phasetransition/dispersion, electrochemical detection, andsemiconductor-based sensing.

17. The method according to any one of 1-16, wherein the single strandeddetector DNA comprises a fluorescence-emitting dye pair.

18. The method according to 17, wherein the fluorescence-emitting dyepair produces an amount of detectable signal prior to cleavage of thesingle stranded detector DNA, and the amount of detectable signal isreduced after cleavage of the single stranded detector DNA.

19. The method according to 17, wherein the single stranded detector DNAproduces a first detectable signal prior to being cleaved and a seconddetectable signal after cleavage of the single stranded detector DNA.

20. The method according to any one of 17-19, wherein thefluorescence-emitting dye pair is a fluorescence resonance energytransfer (FRET) pair.

21. The method according to 17, wherein an amount of detectable signalincreases after cleavage of the single stranded detector DNA.

22. The method according to 17 or 21, wherein the fluorescence-emittingdye pair is a quencher/fluor pair.

23. The method according to any one of 17-22, wherein the singlestranded detector DNA comprises two or more fluorescence-emitting dyepairs.

24. The method according to 23, wherein said two or morefluorescence-emitting dye pairs include a fluorescence resonance energytransfer (FRET) pair and a quencher/fluor pair.

25. The method according to any one of 1-24, wherein the single strandeddetector DNA comprises a modified nucleobase, a modified sugar moiety,and/or a modified nucleic acid linkage.

26. A kit for detecting a target DNA in a sample, the kit comprising:

-   -   (a) a guide RNA, or a nucleic acid encoding the guide RNA, or a        precursor guide RNA array comprising the guide RNA, or a nucleic        acid encoding the precursor guide RNA array; wherein the guide        RNA comprises: a region that binds to a type V CRISPR/Cas        effector protein, and a guide sequence that is complementary to        a target DNA; and    -   (b) a labeled detector DNA that is single stranded and does not        hybridize with the guide sequence of the guide RNA.

27. The kit of 26, further comprising a type V CRISPR/Cas effectorprotein.

28. The kit of 27, wherein the type V CRISPR/Cas effector protein is aCas12 protein.

29. The kit of 27, wherein the type V CRISPR/Cas effector protein is aCas12a (Cpf1) or Cas12b (C2c1) protein.

30. The kit of any one of 26-29, wherein the single stranded detectorDNA comprises a fluorescence-emitting dye pair.

31. The kit of 30, wherein the fluorescence-emitting dye pair is a FRETpair.

32. The kit of 30, wherein the fluorescence-emitting dye pair is aquencher/fluor pair.

33. The kit of any one of 30-32, wherein the single stranded detectorDNA comprises two or more fluorescence-emitting dye pairs.

34. The kit of 33, wherein said two or more fluorescence-emitting dyepairs include a first fluorescence-emitting dye pair that produces afirst detectable signal and a second fluorescence-emitting dye pair thatproduces a second detectable signal.

35. A method of cleaving single stranded DNAs (ssDNAs), the methodcomprising:

-   -   contacting a population of nucleic acids, wherein said        population comprises a target DNA and a plurality of non-target        ssDNAs, with:    -   (i) a type V CRISPR/Cas effector protein; and    -   (ii) a guide RNA comprising: a region that binds to the type V        CRISPR/Cas effector protein, and a guide sequence that        hybridizes with the target DNA,    -   wherein the type V CRISPR/Cas effector protein cleaves        non-target ssDNAs of said plurality.

36. The method of 35, comprising contacting the sample with a precursorguide RNA array, wherein the type V CRISPR/Cas effector protein cleavesthe precursor guide RNA array to produce said guide RNA and at least oneadditional guide RNA.

37. The method of 35 or 36, wherein said contacting is inside of a cellin vitro, ex vivo, or in vivo.

38. The method of 37, wherein the cell is a eukaryotic cell.

39. The method of 38, wherein the eukaryotic cell is a plant cell.

40. The method of any one of 37-39, wherein the non-target ssDNAs areforeign to the cell.

41. The method of 40, wherein the non-target ssDNAs are viral DNAs.

42. The method of any one of 35-41, wherein the target DNA is singlestranded.

43. The method of any one of 35-41, wherein the target DNA is doublestranded.

44. The method of any one of 35-43, wherein the target DNA is viral DNA.

45. The method of any one of 35-43, wherein the target DNA ispapovavirus, hepdnavirus, herpesvirus, adenovirus, poxvirus, orparvovirus DNA.

Set B 1. A method of detecting a target DNA in a sample, the methodcomprising:

-   -   (a) contacting the sample with:        -   (i) a type V CRISPR/Cas effector protein;        -   (ii) a guide RNA comprising: a region that binds to the type            V CRISPR/Cas effector protein, and a guide sequence that            hybridizes with the target DNA; and        -   (iii) a detector DNA that is single stranded and does not            hybridize with the guide sequence of the guide RNA; and    -   (b) measuring a detectable signal produced by cleavage of the        single stranded detector DNA by the type V CRISPR/Cas effector        protein, thereby detecting the target DNA.

2. The method of 1, comprising contacting the sample with a precursorguide RNA array, wherein the type V CRISPR/Cas effector protein cleavesthe precursor guide RNA array to produce said guide RNA and at least oneadditional guide RNA.

3. The method of 1 or 2, wherein the target DNA is single stranded.

4. The method of 1 or 2, wherein the target DNA is double stranded.

5. The method of any one of 1-4, wherein the target DNA is viral DNA.

6. The method of any one of 1-4, wherein the target DNA is papovavirus,hepdnavirus, herpesvirus, adenovirus, poxvirus, or parvovirus DNA.

7. The method of any one of 1-4, wherein the type V CRISPR/Cas effectorprotein is a Cas12 protein.

8. The method of any one of 1-6, wherein the type V CRISPR/Cas effectorprotein is a Cas12a (Cpf1) or Cas12b (C2c1) protein.

9. The method of any one of 1-6, wherein the type V CRISPR/Cas effectorprotein is a Cas12d (CasY) or Cas12e (CasX) protein.

10. The method according to any one of 1-9, wherein the sample comprisesDNA molecules from a cell lysate.

11. The method according to any one of 1-10, wherein the samplecomprises cells.

12. The method according to any one of 1-11, wherein said contacting iscarried out inside of a cell in vitro, ex vivo, or in vivo.

13. The method according to 12, wherein the cell is a eukaryotic cell.

14. The method according to any one of 1-13, wherein the target DNA canbe detected at a concentration as low as 200 fM.

15. The method according to any one of 1-14, comprising determining anamount of the target DNA present in the sample.

16. The method according to 15, wherein said determining comprises:

measuring the detectable signal to generate a test measurement;

measuring a detectable signal produced by a reference sample or cell togenerate a reference measurement; and

comparing the test measurement to the reference measurement to determinean amount of target DNA present in the sample.

17. The method according to any one of 1-16, wherein measuring adetectable signal comprises one or more of: gold nanoparticle baseddetection, fluorescence polarization, colloid phasetransition/dispersion, electrochemical detection, andsemiconductor-based sensing.

18. The method according to any one of 1-17, wherein the single strandeddetector DNA comprises a fluorescence-emitting dye pair.

19. The method according to 18, wherein the fluorescence-emitting dyepair produces an amount of detectable signal prior to cleavage of thesingle stranded detector DNA, and the amount of detectable signal isreduced after cleavage of the single stranded detector DNA.

20. The method according to 18, wherein the single stranded detector DNAproduces a first detectable signal prior to being cleaved and a seconddetectable signal after cleavage of the single stranded detector DNA.

21. The method according to any one of 18-20, wherein thefluorescence-emitting dye pair is a fluorescence resonance energytransfer (FRET) pair.

22. The method according to 18, wherein an amount of detectable signalincreases after cleavage of the single stranded detector DNA.

23. The method according to 18 or 22, wherein the fluorescence-emittingdye pair is a quencher/fluor pair.

24. The method according to any one of 18-23, wherein the singlestranded detector DNA comprises two or more fluorescence-emitting dyepairs.

25. The method according to 24, wherein said two or morefluorescence-emitting dye pairs include a fluorescence resonance energytransfer (FRET) pair and a quencher/fluor pair.

26. The method according to any one of 1-25, wherein the single strandeddetector DNA comprises a modified nucleobase, a modified sugar moiety,and/or a modified nucleic acid linkage.

27. The method according to any one of 1-26, wherein the methodcomprises amplifying nucleic acids in the sample.

28. The method according to 27, wherein said amplifying comprisesisothermal amplification.

29. The method according to 28, wherein the isothermal amplificationcomprises recombinase polymerase amplification (RPA).

30. The method according to any one of 27-29, wherein said amplifyingbegins prior to the contacting of step (a).

31. The method according to any one of 27-29, wherein said amplifyingbegins together with the contacting of step (a).

32. A kit for detecting a target DNA in a sample, the kit comprising:

-   -   (a) a guide RNA, or a nucleic acid encoding the guide RNA, or a        precursor guide RNA array comprising the guide RNA, or a nucleic        acid encoding the precursor guide RNA array; wherein the guide        RNA comprises: a region that binds to a type V CRISPR/Cas        effector protein, and a guide sequence that is complementary to        a target DNA; and    -   (b) a labeled detector DNA that is single stranded and does not        hybridize with the guide sequence of the guide RNA.

33. The kit of 32, further comprising a type V CRISPR/Cas effectorprotein.

34. The kit of 33, wherein the type V CRISPR/Cas effector protein is aCas12 protein.

35. The kit of 33, wherein the type V CRISPR/Cas effector protein is aCas12a (Cpf1) or Cas12b (C2c1) protein.

36. The kit of 33, wherein the type V CRISPR/Cas effector protein is aCas12d (CasY) or Cas12e (CasX) protein.

37. The kit of any one of 32-36, wherein the single stranded detectorDNA comprises a fluorescence-emitting dye pair.

38. The kit of 37, wherein the fluorescence-emitting dye pair is a FRETpair.

39. The kit of 37, wherein the fluorescence-emitting dye pair is aquencher/fluor pair.

40. The kit of any one of 37-39, wherein the single stranded detectorDNA comprises two or more fluorescence-emitting dye pairs.

41. The kit of 40, wherein said two or more fluorescence-emitting dyepairs include a first fluorescence-emitting dye pair that produces afirst detectable signal and a second fluorescence-emitting dye pair thatproduces a second detectable signal.

42. The kit of any one of 32-41, further comprising nucleic acidamplification components.

43. The kit of 42, wherein the nucleic acid amplification components arecomponents for recombinase polymerase amplification (RPA).

44. A method of cleaving single stranded DNAs (ssDNAs), the methodcomprising:

contacting a population of nucleic acids, wherein said populationcomprises a target DNA and a plurality of non-target ssDNAs, with:

-   -   (i) a type V CRISPR/Cas effector protein; and    -   (ii) a guide RNA comprising: a region that binds to the type V        CRISPR/Cas effector protein, and a guide sequence that        hybridizes with the target DNA,

wherein the type V CRISPR/Cas effector protein cleaves non-target ssDNAsof said plurality.

45. The method of 44, comprising contacting the sample with a precursorguide RNA array, wherein the type V CRISPR/Cas effector protein cleavesthe precursor guide RNA array to produce said guide RNA and at least oneadditional guide RNA.

46. The method of 44 or 45, wherein said contacting is inside of a cellin vitro, ex vivo, or in vivo.

47. The method of 46, wherein the cell is a eukaryotic cell.

48. The method of 47, wherein the eukaryotic cell is a plant cell.

49. The method of any one of 46-48, wherein the non-target ssDNAs areforeign to the cell.

50. The method of 49, wherein the non-target ssDNAs are viral DNAs.

51. The method of any one of 44-50, wherein the target DNA is singlestranded.

52. The method of any one of 44-50, wherein the target DNA is doublestranded.

53. The method of any one of 44-52, wherein the target DNA is viral DNA.

54. The method of any one of 44-52, wherein the target DNA ispapovavirus, hepdnavirus, herpesvirus, adenovirus, poxvirus, orparvovirus DNA.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1

FIG. 3. Non-complementary strand cleavage is dictated by complementarystrand recognition. The length of the non-target strand (NTS) (top gel)or target strand (TS) (bottom gel) was altered to determine thesubstrate requirements for Cas12 cleavage. LbCas12a-crRNA complexes arein large excess over radiolabeled substrates, and cleavage products areresolved by denaturing polyacrylamide gel electrophoresis (PAGE). The TSwas trimmed to single nucleotides regardless of the length of the NTS,whereas the NTS was cleaved only when at least 15 nt of complementary TSis present.

FIG. 4. Complementary strand binding unleashes non-specific DNaseactivity by Cas12a. It was tested whether a non-complementary, randomssDNA is prone to degradation upon Cas12a activation by a complementarytarget strand. LbCas12a-crRNA complexes are in large excess overradiolabeled substrates, and cleavage products are resolved bydenaturing PAGE. The random ssDNA radiolabeled target (blue) wasdegraded only when LbCas12a was pre-complexed with an “activator”complementary target strand. The random dsDNA radiolabeled target (blue)was protected from cleavage.

FIG. 5. The non-target strand was cleaved only in the presence of atarget strand with at least 15 nt of complementarity. LbCpf1 cleavedtarget strand DNA regardless of the length of the non-target strand.LbCpf1 cleaved non-target strand DNA only when the target strand had atleast 15 nt of complementarity.

FIG. 6. The RuvC nuclease is responsible for trans-cleavage of ssDNA. Inthe presence of the activator target strand, non-specific trans-cleavagewas not observed with a catalytically-inactive RuvC nuclease (pXT002).Trans-cleavage was still observed with a RNA-processing dead mutant(pXT006).

FIG. 7. Targeting by two homologs of Cas12a results in rapid “shredding”of M13 phage ssDNA. It was tested whether free 5′ or 3′ ends wererequired for trans-cleavage by using M13 phage circular ssDNA as a transsubstrate. LbCas12a-crRNA and AsCas12a-crRNA was pre-complexed with assDNA activator (with no sequence complementarity to M13 phage) andincubated with M13 ssDNA at 37 C; products were resolved on a 1.5%agarose gel and visualized with SyberGold. Rapid shredding was observedat the earliest time point (1 min), and was both activator- andRuvC-dependent. The same trend with AsCas12a was observed suggestingthat this activity is likely conserved across Cas12a homologs.

FIG. 8. Trans-cleavage by Cas12a can be detected using an FQ-basedassay. To improve the throughput of measuring trans-cleavage, anFQ-based assay using a DNase Alert substrate (IDT) was adapted as aprobe for trans-activation. A fluorescence signal was released uponcleavage of the substrate, which contained a DNA linker and neighboringquencher.

FIG. 9. Trans-activation is sensitive to mismatches at the PAM-proximalend with duplexed substrates, but not with single-stranded substrates.Using the FQ-based assay, the mismatch tolerance for activatingtrans-cleavage was tested. LbCas12a-crRNA was pre-complexed with eitherssDNA or dsDNA containing 2bp mismatches from the PAM-distal toPAM-proximal end, or a mutated PAM. The top panel showsbackground-subtracted max fluorescence after lh incubation at 37 C forssDNA (left) or dsDNA (right). The bottom panel shows observed cleavagerates after lh incubation at 37 C. Mismatches appeared highly toleratedin the case of ssDNA substrates, but PAM-proximal mismatches were poorlytolerated in the case of dsDNA substrates likely due to inadequate RNAstrand invasion. Notably, trans-cleavage was PAM-independent when Cas12ais activated by ssDNA.

FIG. 10. dsDNA target (cis) cleavage follows single-turnover kinetics,whereas ssDNA (trans) cleavage is multiple-turnover. Turnover kineticswere assayed for cis and trans cleavage by incubating radiolabeled dsDNA(left) or random ssDNA (right) with respective LbCas12a-crRNA ratios.Each point represents quantified % cleavage after a 30 minute incubationwith LbCas12a at 37 C (via denaturing PAGE). For the ssDNA kinetics,LbCas12a-crRNA was pre-complexed with ssDNA activator before addition ofradiolabeled random ssDNA. The data show that cis cleavage by LbCas12ais single-turnover, whereas trans cleavage is multiple turnover.

FIG. 11. Trans-ssDNA cleavage by Cas12a can be harnessed as a simplediagnostic to distinguish viral serotypes such as HPV and otherclinically relevant DNA viruses. Cas12a can detect targets as low aspicomolar concentrations. To demonstrate that LbCas12a trans-activitycan be harnessed as a simple diagnostic, the FQ-based assay using DNaseAlert substrate was used to test whether one could distinguish twoclosely-related HPV sequences (HPV16 and HPV18) that are consideredhigh-risk strains for cervical cancer. LbCas12 is pre-incubated with acrRNA targeting a HPV16 and HPV18 sequence adjacent to a TTTA PAM; thetwo sequences differ by 6 nucleotides. 500 bp fragments of HPV16 andHPV18 were cloned into a plasmid backbone (˜6kb total) as a proxy forthe full HPV genome (˜8kb), and incubated with LbCas12a-crRNA for 30 min(top) or lh (bottom) at 37 C. HPV serotypes were easily distinguishedand the method could detect down to ˜10 pM of target. This method couldin principle be extended to detect any DNA virus, and examples ofclinically-relevant DNA viruses are listed herein.

FIG. 12. A unifying model for DNA cleavage by CRISPR-Cas12a Cas12a-crRNAcomplex binds to a substrate in PAM-dependent (dsDNA) or PAM-independent(ssDNA) manner When the dsDNA PAM is recognized, the duplex isinterrogated by RNA strand invasion and recognition of the complementarytarget strand activates the RuvC nuclease to cleave both the unwound TSand NTS. Binding of the complementary ssDNA also triggers the RuvCnuclease to degrade any ssDNAs.

Example 2

CRISPR-Cas12a (Cpf1) belongs to a family of RNA-guided DNA targetingenzymes that bind and cut DNA as components of bacterial adaptive immunesystems. Like CRISPR-Cas9, Cas12a and related enzymes are also powerfulgenome editing tools based on their ability to induce genetic changes incells at sites of double-stranded DNA cuts. In the course ofinvestigating the DNA substrate selectivity of Cas12a, the inventorswere surprised to find that RNA-guided DNA binding unleashes robust,non-specific single-stranded DNA (ssDNA) cleavage activity sufficient tocompletely degrade both linear and circular ssDNA molecules. Thisactivity, catalyzed by the same active site responsible forsite-specific dsDNA cutting, shreded ssDNA irrespective of sequencerequirements and with rapid multiple-turnover cleavage kinetics.Activation of ssDNA cutting required faithful recognition of a DNAtarget sequence matching the guide sequence of the guide RNA withspecificity sufficient to distinguish between closely related viralserotypes. The data provided herein show that Cas12a-catalyzed ssDNAdegradation, not observed for CRISPR-Cas9 enzymes, is a fundamentalproperty of other Cas12-family proteins, revealing a fascinating andsurprising parallel with the RNA-triggered general RNase activity of thetype VI CRISPR-Cas13 enzymes.

Results

CRISPR-Cas adaptive immunity in bacteria and archaea uses RNA-guidednucleases to identify and cut foreign nucleic acids. The CRISPR-Cas9family of enzymes has been widely deployed for gene editing applicationsin eukaryotes based on the precision of double-stranded DNA (dsDNA)cleavage induced by two catalytic domains, RuvC and HNH, at sequencescomplementary to a guide RNA sequence. A second family of enzymesharnessed for gene editing, CRISPR-Cas12a (formerly known as Cpf1), usesa single catalytic domain (RuvC) for guide RNA-directed dsDNA cleavage(FIG. 13a ). Distinct from Cas9, Cas12a enzymes also process individualguide RNAs from a longer precursor transcript and generate dsDNA breakswith staggered 5′ and 3′ ends, features that have attracted interest inCas12a for gene editing applications. Despite its adoption as agenome-editing tool, the substrate specificity and DNA cleavagemechanism of Cas12a are yet to be fully elucidated.

While the DNA substrate requirements for Cas12a activation were beinginvestigated, Lachnospiraceae bacterium ND2006 Cas12a (LbaCas12a) wastested for guide RNA-directed single-stranded DNA (ssDNA) cleavage, acapability of various CRISPR-Cas9 orthologs. Purified LbaCas12a orSpyCas9 proteins were assembled with guide RNAs that have base pairingcomplementarity to circular, single-stranded M13 DNA phage. AlthoughSpyCas9 catalyzed site-specific M13 cleavage, generating linear phagemolecules as expected, LbaCas12a surprisingly induced rapid and completedegradation of M13 by a cleavage mechanism that could not be explainedby sequence-specific DNA cutting (FIG. 13b ). This robust ssDNAdegradation was not observed in experiments using an LbaCas12a proteincontaining inactivating mutations in the RuvC catalytic domain. Theseresults suggested that LbaCas12a possesses a unique ssDNA shreddingactivity that requires the same active site used for RNA-directed dsDNAcutting.

The non-target strand (NTS) was cleaved only when the target strand (TS)contained at least 15 nt of complementarity with the guide RNA (FIG.17). These results suggested that TS recognition is a prerequisite forssDNA cutting, raising the possibility that LbaCas12a possessesnon-specific ssDNase activity. To test the idea that a TS-activatedLbaCas12a could cut any ssDNA, LbaCas12a was pre-complexed with a crRNAand complementary ssDNA or dsDNA activator, and introduced an unrelatedradiolabeled ssDNA, dsDNA or ssRNA in trans. Remarkably, both ssDNA anddsDNA activators triggered LbaCas12a to completely degrade the ssDNAtrans-substrate to its 5′end label in a RuvC-dependent manner (FIG. 18,FIG. 19, FIG. 20), whereas the dsDNA and ssRNA trans-substrates remainedprotected from the activated complex (FIG. 21). Together, these findingsrevealed that Cas12a DNA binding unleashes robust, non-specific ssDNasetrans-activity by the RuvC nuclease.

The rapid degradation of a trans substrate suggested that the kineticsof non-specific ssDNA trans-cleavage may be fundamentally different fromcis-cleavage, in which LbaCas12a targets a complementary dsDNAsubstrate. To investigate how a single RuvC nuclease cuts by twodifferent mechanisms, substrate turnover was observed by titrating molarratios of either LbaCas12a-crRNA or LbaCas12a-crRNA-ssDNA activatorcomplexes against a dsDNA target (cis) or non-specific ssDNA (trans)substrate, respectively. The fraction of cleaved target dsDNA wasproportional to the molar ratio of LbaCas12a-crRNA to DNA, demonstratingthat cis-cleavage is single-turnover (FIG. 14a ). In contrast, thefraction of cleaved non-specific ssDNA was saturated at sub-equimolarratios, revealing that trans-cleavage follows multiple turnover kinetics(FIG. 14b ). To further examine the Michaelis-Menten kinetics oftrans-cleavage, a real-time, fluorophore quencher (FQ)-labeled DNAreporter assay was adapted to measure non-specific DNase activity underconditions where LbaCas12a-crRNA is stably bound to a ssDNA or dsDNAactivator. LbaCas12a pre-complexed with a ssDNA activator revealed ahighly robust activity that yielded a catalytic efficiency(k_(cat)/K_(m)) of 5.1×10⁸ s⁻¹ M⁻¹. When pre-complexed with a dsDNAactivator, the catalytic efficiency was nearly an order of magnitudefaster and approached the rate of diffusion with a k_(cat)/K_(m)measurement of 1.7×10⁹ s⁻¹ M⁻¹ (FIG. 14c , FIG. 22). These differencesin catalytic efficiencies suggest a potential role for the NTS of thedsDNA activator to stabilize the Cas12a complex in an optimalconformation for cutting a trans-ssDNA substrate.

The substrate specificity of a ssDNA versus dsDNA activator fortrans-cleavage was next considered. First, experiments were performed toconfirm that PAM recognition is critical for activation by acomplementary dsDNA but not for a matching ssDNA, consistent with therequirements for target binding (FIG. 15a ). To test whether mismatchesalong the activator sequence could impact the rate of trans-cleavage,two base-pair (bp) mismatches were introduced across the target sequencein either a ssDNA or dsDNA activator. Using the FQ-based assay,LbaCas12a was pre-loaded with the crRNA and activator before addition ofthe ssDNA reporter, and the real-time increase in fluorescence signalwas measured as a proxy for the observed trans-cleavage rate. Whereasmismatches across the ssDNA activator sequence were generally welltolerated, mismatches in the PAM or “seed region” of the dsDNA activatorwere poorly tolerated (FIG. 15b , FIG. 23). These trends using a dsDNAactivator suggest that PAM recognition and unidirectional DNA unwindingprovide additional regulation for trans-cleavage. However, extensivebase complementary between the crRNA and target strand is the onlyrequirement for activating trans-cleavage.

Because LbaCas12a demonstrated higher specificity using dsDNA activatorsfor trans-cleavage, the FQ-based assay was used to test whetherLbaCas12a could be readily programmed to distinguish between twoclosely-related dsDNA viruses. As a proof-of-principle, the Humanpapillomavirus (HPV) serotypes 16 (HPV16) and 18 (HPV18) were selected,which account for approximately 70% of all cases of cervical cancerfollowing persistent HPV infection. LbaCas12a was first pre-complexedwith a crRNA targeting an HPV16 or HPV18 sequence adjacent to a TTTA PAMthat differ by only 6 nucleotides (FIG. 15c ). As a proxy for the fullHPV genome (˜8kb), 500 bp fragments of HPV16 and HPV18 were cloned intoa ˜5kb plasmid, and incubated the HPV-containing plasmid withLbaCas12a-crRNA. Robust activation of trans-cleavage was observed onlywhen LbaCas12a was in the presence of at least ˜10 pM of its cognate HPVtarget (FIG. 15d , FIG. 24), suggesting that the native specificity ofdsDNA recognition and trans-cleavage activation by LbaCas12a could inprinciple be extended to detect any dsDNA virus.

It was then tested whether this trans-cleavage activity might beconserved among the Cas12a family, and even more broadly acrossevolutionarily distinct type V effector proteins. Two lines of evidencehinted at this possibility: first, target-bound crystal structures ofCas12b (previously known as C2c1) suggested that its RuvC catalyticpocket accommodates both the TS and the NTS for cleavage, similar to thecis-cleavage mechanism proposed for Cas12a. Second, despite low sequenceand structural similarity between these subtypes, a unifying structuralfeature among all Cas12 proteins is the RuvC nuclease domain near theC-terminal end of the polypeptide. Therefore, two additional Cas12aorthologs from Acidaminococcus sp. (AspCas12a) and Francisella novicida(FnoCas12a) were selected, as well as a Cas12b protein fromAlicyclobacillus acidoterrestris (AacCas12b) to test for cis- andtrans-cleavage (FIG. 16a ). Despite varying efficiencies, all of thehomologs evaluated demonstrated non-specific ssDNase activity whenpre-complexed with a complementary ssDNA activator (FIG. 16b ),suggesting that trans-cleavage is a fundamental property of Cas12-familyproteins. These experiments further underscore the functionalconvergence of trans-cleavage between the DNA-targeting type V andRNA-targeting type VI effector proteins.

The data herein suggest a new mechanism for target interference by theCas12 protein family and a new model is proposed herein in which theCas12-guide RNA complex binds to a DNA substrate in a PAM-dependent(dsDNA) or PAM-independent (ssDNA) manner (FIG. 16c ). Following PAMrecognition for a dsDNA substrate, RNA strand invasion and targetrecognition activates the RuvC nuclease to cleave the unwound TS andtrim back the NTS, thereby generating the staggered dsDNA break androbustly activating ssDNA trans-cleavage. Binding of a complementaryssDNA bypasses PAM recognition and RNA strand invasion, but issufficient to trigger the RuvC nuclease to degrade any ssDNAs.

Example 3 CRISPR-Cas12a Target Binding Unleashes IndiscriminateSingle-Stranded DNase Activity

The data presented here show that RNA-guided DNA binding unleashesrobust, indiscriminate single-stranded DNA (ssDNA) cleavage activity inCas12 proteins (e.g., Cas12a) sufficient to completely degrade bothlinear and circular ssDNA molecules. The data show that target-activatednon-specific ssDNase activity, catalyzed by the same active siteresponsible for site-specific dsDNA cutting, is a fundamental propertyof type V CRISPR-Cas12 enzymes. Activation of ssDNA cutting requiresfaithful recognition of a DNA target sequence matching the guidesequence of the guide RNA with specificity capable of distinguishingclosely related DNA sequences. Target-dependent Cas12 ssDNase activationwas combined with isothermal amplification to create a method termed DNAEndonuclease Targeted CRISPR Trans Reporter (DETECTR), which achievedattomolar sensitivity for nucleic acid detection. DETECTR isdemonstrated here to facilitate rapid and specific detection of DNA(e.g., HPV) in human patient samples, thereby providing a simpleplatform for nucleic acid-based, point-of-care diagnostics.

CRISPR-Cas adaptive immunity in bacteria and archaea uses RNA-guidednucleases to target and degrade foreign nucleic acids. The CRISPR-Cas9family of proteins has been widely deployed for gene editingapplications based on the precision of double-stranded DNA (dsDNA)cleavage induced by two catalytic domains, RuvC and HNH, at sequencescomplementary to a guide RNA sequence. A second family of enzymes,CRISPR-Cas12a (Cpf1), uses a single RuvC catalytic domain for guideRNA-directed dsDNA cleavage (FIG. 25A). Distinct from Cas9, Cas12aenzymes recognize a T-rich protospacer adjacent motif (PAM), catalyzetheir own guide RNA (crRNA) maturation and generate a PAM-distal dsDNAbreak with staggered 5′ and 3′ ends, features that have attractedinterest for gene editing applications.

While investigating substrate requirements for Cas12a activation,Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) was tested for guideRNA-directed single-stranded DNA (ssDNA) cleavage, a capability ofdiverse CRISPR-Cas9 orthologs. Purified LbCas12a or Streptococcuspyogenes Cas9 (SpCas9) proteins (FIG. 30) were assembled with guide RNAsequences targeting a circular, single-stranded M13 DNA phage. Incontrast to SpCas9, it was surprising to find that LbCas12a inducedrapid and complete degradation of M13 by a cleavage mechanism that couldnot be explained by sequence-specific DNA cutting (FIG. 25B). This ssDNAshredding activity, not observed using an LbCas12a protein containing aninactivating mutation in the RuvC catalytic domain (D832A), raised thepossibility that a target-bound LbCas12a could degrade any ssDNA,regardless of complementarity to the guide RNA. To test this idea,LbCas12a or SpCas9 was assembled with a different guide RNA and itscomplementary ssDNA that has no sequence homology to M13 phage genomesequence, and single-stranded M13 DNA was added to the reaction.Remarkably, LbCas12a catalyzed M13 degradation only in the presence ofthis complementary ssDNA “activator”, an activity not observed forSpCas9 (FIG. 25C). These findings revealed that binding of theLbCas12a-crRNA complex to a guide-complementary ssDNA unleashed robust,non-specific ssDNA trans-cleavage activity.

FIG. 25. Cas12a target recognition activated non-specificsingle-stranded DNA cleavage. (A) Cas12a-crRNA complex binds a dsDNAsubstrate and generates a 5′ overhang staggered cut using a single RuvCnuclease. (B, C) Representative M13 ssDNA cleavage timecourses withpurified LbCas12a (left) and SpCas9 (right) complexed with a (B) guideRNA complementary to M13 phage or (C) a guide RNA and complementaryssDNA activator with no sequence homology to M13 phage.

FIG. 30. Purification of Cas12 and Cas9 proteins. SDS-PAGE gel of allpurified Cas12 and Cas9 proteins used in this study.

The requirements for LbCas12a-catalyzed trans-cleavage activity was nextinvestigated. Using a fluorophore quencher (FQ)-labeled reporter assay,LbCas12a was assembled with its crRNA and either a complementary ssDNA,dsDNA or single-stranded RNA (ssRNA), and an unrelated ssDNA- orssRNA-FQ reporter was introduced in trans (FIG. 31). Both thecrRNA-complementary ssDNA or dsDNA (the activator) triggered LbCas12a tocleave the ssDNA-FQ reporter substrate (FIG. 31A). However, ssRNA wasneither capable of activating trans-cleavage nor susceptible todegradation by LbCas12a (FIG. 31B), confirming that LbCas12a harbors aDNA-activated general DNase activity.

FIG. 31. LbCas12a is a DNA-activated general DNase. Quantification ofmaximum fluorescence signal generated after incubatingLbCas12a-crRNA-activator with a custom (A) trans-ssDNA-FQ or (B)trans-ssRNA-FQ reporter for lh at 37° C., with DNase I or RNase Acontrols where indicated. Error bars represent the mean ±s.d., where n=3replicates.

To determine how LbCas12a-catalyzed ssDNA cleavage activity relates tosite-specific dsDNA cutting, the length requirements of both the targetstrand (TS) and non-target strand (NTS) for LbCas12a activation wastested using radiolabeled oligonucleotides. Although TS cutting occurredirrespective of the NTS length (FIG. 32A), NTS cleavage occurred onlywhen the TS contained at least 15 nucleotides (nt) of complementaritywith the crRNA (FIG. 32B). This showed that TS recognition is aprerequisite for NTS cutting. To test whether LbCas12a remains activefor non-specific ssDNA cleavage after sequence-specific binding andcleavage of a dsDNA substrate, a dsDNA plasmid was first cut with anLbCas12a-crRNA complex, and then an unrelated dsDNA or ssDNA was addedto the reaction (FIG. 26A). Whereas the non-specific dsDNA substrateremained intact, the ssDNA was rapidly degraded in a RuvC-domaindependent manner (FIG. 26A; FIG. 33; FIG. 34). Using truncatedactivators that are too short to be cleaved, it was next determined thatonly target DNA binding is required to activate trans-ssDNA cleavage(FIG. 35). Together, these results show that RNA-guided DNA bindingactivates LbCas12a for both site-specific dsDNA cutting and non-specificssDNA trans-cleavage.

FIG. 26. Kinetics of Cas12a ssDNA trans-cleavage. (A) Sequence-specificplasmid DNA cleavage reactions by LbCas12a-crRNA (top) were introducedto a separate radiolabeled dsDNA or ssDNA substrate of unrelatedsequence (bottom); timecourses represent minutes. (B) Target dsDNA or(C) non-specific ssDNA incubated with molar ratios of LbCas12a-crRNA asindicated. Each point represents the mean quantified percent cleavageafter 30 minutes at 37° C., at which time the reaction was atcompletion. Error bars represent the mean ±s.d., where n=3 replicates.(D) Representative Michaelis-Menten plot for LbCas12a-catalyzed ssDNAtrans-cleavage using a dsDNA or ssDNA activator. Measured k_(cat)/K_(m)values report mean ±s.d., where n=3 replicates.

FIG. 32. Target strand recognition is a pre-requisite forsingle-stranded DNA cleavage. Cleavage timecourse assays using LbCas12awith (A) truncated non-target strand (NTS) annealed to a radiolabeledtarget strand (TS), (B) truncated TS annealed to a radiolabeled NTS.Timecourses represent minutes and cleavage products resolved bydenaturing PAGE. Schematic on right depicts cleavage of the radiolabeledTS (A) or NTS (B), which generates a Cas12a-mediated staggered cut.

FIG. 33. The RuvC nuclease domain is responsible foractivator-dependent, non-specific DNase activity. Cleavage timecoursegel with radiolabeled non-target strand of a complementary dsDNA andnon-specific ssDNA substrate using (A) WT LbCas12a, (B) RuvC catalyticmutant (D832A) and (C) crRNA-processing mutant (H759A), with or withouta ssDNA activator. Timecourses represent minutes and cleavage productswere resolved by denaturing PAGE.

FIG. 34. LbCas12a trans-cleavage degrades complementary and non-specificssDNA, but not ssRNA. Cleavage timecourse gels of LbCas12a-crRNAcomplexes using (A) no activator, (B) ssDNA activator in 1.2-fold molarexcess, or (C) ssDNA activator in 100-fold molar excess. Radiolabeledsubstrates are indicated, where cis indicates a complementary target andtrans indicates a non-complementary sequence. For cis substrates, thenon-target strand was radiolabeled. Timecourses represent minutes andcleavage products were resolved by denaturing PAGE.

FIG. 35. Target strand cleavage by Cas12a is not required for triggeringnon-specific ssDNase activity. Cleavage timecourse assays using LbCas12awith (A) radiolabeled target strand with either a ssDNA (10-25 nt) ordsDNA (10-25 bp) substrate, or (B) radiolabeled non-specific ssDNAsubstrate in the presence of either a ssDNA (10-25 nt) or dsDNA (10-25bp) activator. Timecourses represent minutes and cleavage products wereresolved by denaturing PAGE.

The rapid degradation of a trans substrate suggested that the kineticsof LbCas12a-catalyzed site-specific dsDNA (cis-) cleavage andnon-specific ssDNA (trans-) cleavage are fundamentally different.Stoichiometric titration experiments showed that cis-cleavage issingle-turnover (FIG. 26B), whereas trans-cleavage is multiple-turnover(FIG. 26C). Although the Cas12a-crRNA complex remains bound to the dsDNAtarget following cis-cleavage, the complex releases its PAM-distalcleavage products from the RuvC active site, enabling ssDNA substrateaccess and turnover. Using the FQ assay, it was found thatLbCas12a-crRNA bound to a ssDNA activator molecule catalyzed trans-ssDNAcleavage at a rate of ˜250 per second and a catalytic efficiency(k_(cat)/K_(m)) of 5.1×10⁸ s M′. When bound to a dsDNA activator,LbCas12a-crRNA catalyzed ˜1250 turnovers per second with a catalyticefficiency approaching the rate of diffusion with a k_(cat)/K_(m) of1.7×10⁹ s⁻¹ M⁻¹ (FIG. 26D; FIG. 36). These differences suggested thatthe NTS of the dsDNA activator helps stabilize the Cas12a complex in anoptimal conformation for trans-ssDNA cutting.

FIG. 36. Michaelis-Menten analysis revealed robust trans-cleavageactivity with a ssDNA and dsDNA activator. Representative plots ofinitial velocity versus time for a (A) ssDNA or (C) dsDNA activator,using 0.1 nM effective LbCas12a-crRNA-activator complex and increasingDNaseAlert substrate concentrations at 37° C. Michaelis-Menten fits forthe corresponding (B) ssDNA or (D) dsDNA activator. (E) Calculatedk_(cat), K_(m) and k_(cat)/K_(m) values report the mean ±s.d., where n=3replicates.

The specificity of trans-cleavage activation was next tested usingeither a ssDNA or dsDNA activator. The PAM sequence required for dsDNAbinding by CRISPR-Cas12a was found to be critical for catalyticactivation by a crRNA-complementary dsDNA, but not for acrRNA-complementary ssDNA (FIG. 27A). Two base-pair (bp) mismatchesintroduced along the crRNA-complementary sequence of either a ssDNA ordsDNA activator molecule slowed the trans-cleavage rate of a ssDNA-FQreporter by up to ˜100 fold, depending on the mismatch position. Foronly the dsDNA activator, alterations to the PAM sequence or mismatchesbetween the crRNA and PAM-adjacent “seed region” also had largeinhibitory effects on trans-ssDNA cleavage activity (FIG. 27B; FIG. 37),similar to the mismatch tolerance pattern observed in Cas12a off-targetstudies. Together, these data are consistent with PAM-mediated dsDNAtarget binding and the role of base pairing between the crRNA and thetarget strand to activate trans-ssDNA cutting.

FIG. 27. Specificity and conservation of trans-cleavage activation. (A)LbCas12a-crRNA in the absence or presence of indicated activator,incubated with a radiolabeled non-specific ssDNA substrate (S) for 30min at 37° C.; products (P) resolved by denaturing PAGE. (B) Observedtrans-cleavage rates for LbCas12a using a ssDNA or dsDNA activator withindicated mismatches; rates represent the average of three differenttargets measured in triplicate, and error bars represent mean ±s.d.,where n=9 (three replicates for three independent targets). (C)Radiolabeled cis (complementary) or trans (non-complementary) substrateswere incubated with Cas12a-crRNA or Cas9-sgRNA in the presence orabsence of a ssDNA activator for 30 min at 37° C.; a cis-dsDNA substratewas used in the “no enzyme” lanes. Substrate (S) and nucleotide products(P) were resolved by denaturing PAGE.

FIG. 37. The PAM sequence and PAM-proximal mismatches in a dsDNAactivator provided specificity for trans-activation. Quantification oftrans-cleavage kinetics using mismatched substrates for three distincttarget sequences; error bars represent the mean ±s.d., where n=3replicates.

The data suggested that this trans-ssDNA cutting activity might be aproperty shared by other Cas12a enzymes, and perhaps more evolutionarilydistinct type V CRISPR effector proteins, considering that all type Veffectors contain a single RuvC nuclease domain Consistent with thispossibility, purified Cas12a orthologs from Acidaminococcus sp.(AsCas12a) and Francisella novicida (FnCas12a), as well as a Cas12bprotein from Alicyclobacillus acidoterrestris (AaCas12b), all catalyzednon-specific ssDNase cleavage when assembled with crRNA and acomplementary ssDNA activator (FIG. 27C; FIG. 38). In contrast, none ofthe type II CRISPR-Cas9 proteins tested showed evidence for trans-ssDNAcleavage (FIG. 27C; FIG. 38), suggesting that target-dependentactivation of non-specific ssDNA cleavage is a fundamental feature ofall type V CRISPR-Cas12 proteins. These results reveal the unexpectedfunctional convergence of Cas12 enzymes with the type III CRISPR-Csm/Cmrand type VI CRISPR-Cas13 effectors, which also exhibit target-activated,non-specific ssDNase or ssRNase activity, respectively.

FIG. 38. Activator-dependent, non-specific ssDNA cleavage activity wasfound to be conserved across type V CRISPR interference proteins.Radiolabeled cis (complementary) or trans (non-complementary) substrateswere incubated with Cas12-crRNA in the presence or absence of a ssDNAactivator for 30 min at 37° C. (or 47.5° C. for AaCas12b). Forcis-dsDNA, non-target strand is 5′ end labeled, while the target strand(complementary to guide RNA) is 5′ end labeled for cis-ssDNA;trans-ssDNA and dsDNA are non-specific DNAs. In “no enzyme” lanes, 5′end labeled trans-ssDNA is loaded. Substrate (S) and nucleotide products(P) are resolved by denaturing PAGE.

It was next tested whether LbCas12a could be repurposed as a DNAdetection platform for use in clinical specimens, based on its abilityto induce a fluorescent readout in response to a specific dsDNAsequence. In particular, accurate and rapid identification of humanpapillomavirus (HPV) is critical for identification of those at risk ofHPV-related pre-cancer and cancer, with types 16 (HPV16) and 18 (HPV18)accounting for the majority of precancerous lesions. To test ifLbCas12a-catalyzed trans-ssDNA cleavage can distinguish between thesetwo dsDNA viruses, a 20 nt target sequence located next to a TTTA PAMthat varied by only six base pairs between the two HPV genotypes wasselected (FIG. 39). Plasmids containing a ˜500 bp fragment of the HPV16or HPV18 genome, including the target sequence, were incubated with theLbCas12a-crRNA complex targeting either the HPV16 or HPV18 fragment anda quenched-fluorescent ssDNA reporter. After one hour, LbCas12a produceda robust fluorescent signal only in the presence of the cognate HPVtarget, whose identity could be distinguished down to ˜10 pM of plasmid(FIG. 39). To enhance assay sensitivity, isothermal amplification byRecombinase Polymerase Amplification (RPA) was coupled with LbCas12a todevelop a rapid one-pot detection method termed DNA EndonucleaseTargeted CRISPR Trans Reporter (DETECTR) (FIG. 40A). When programmed torecognize its cognate plasmid, DETECTR was able to identify targets withattomolar sensitivity (FIG. 40B).

FIG. 39. Cas12a distinguishes two closely related HPV sequences. (A)Alignment of 20nt targeting sequences within HPV16 and HPV18 genomesthat differ by 6 nucleotides, with a schematic of Cas12a detection usinga ssDNA-FQ reporter. Fluorescence timecourses with LbCas12a preassembledwith a crRNA targeting (B) HPV16 or (C) HPV16 in the presence of a dsDNAplasmid containing an HPV16 (top row) or HPV18 (middle row) genomicfragment and DNaseAlert substrate, with fluorescence measurements takenevery 30 seconds for 1 h at 37° C. (D) Maximum fluorescence signalobtained from timecourses in (B) and (C). Error bars represent mean±s.d., where n=3 replicates.

FIG. 40. Isothermal amplification coupled with Cas12a detection yieldedDETECTR, which achieved attomolar sensitivity. (A) Schematic of DETECTR,consisting of isothermal amplification by RPA and Cas12a detection usinga ssDNA-FQ reporter. (B) Titration of two independent plasmids detectedby DETECTR or Cas12a alone. Note that DETECTR achievee attomolarsensitivity. Error bars represent mean ±s.d., where n=3 replicates.

To assess whether HPV could be detected in more complex mixtures, DNAextracted from cultured human cells infected with HPV types 16 (SiHa),18 (HeLa), or without HPV (BJAB) was added to LbCas12a complexed with acrRNA targeting the hypervariable loop V of the L1 gene within HPV16 orHPV18 (FIG. 28A).

Whereas LbCas12a-crRNA alone was not sensitive enough to detect HPV,DETECTR unambiguously identified HPV types 16 and 18 only in SiHa andHeLa cells, respectively (FIG. 28B; FIG. 41A, B). To investigate theutility of DETECTR on patient samples, crude DNA extractions from 25human anal swabs previously analyzed by a PCR-based method for HPVinfection were tested (FIG. 42). Within one hour, DETECTR accuratelyidentified the presence or absence of HPV16 (25/25 agreement) and HPV18(23/25 agreement) in 25 patient samples containing a heterogeneousmixture of HPV types, with good correlation between the PCR-basedintensity and DETECTR signal (FIG. 28C, D; FIG. 41C, D; FIG. 42).Furthermore, the absence of fluorescence signal in specimens that werenot infected with HPV types 16 or 18, but did contain other HPV types,was an indicator of good specificity by DETECTR. These resultsdemonstrate a new platform for CRISPR-based diagnostics, and suggestthat DETECTR could in principle be extended to rapidly detect any DNAsequence of interest with high sensitivity and specificity.

FIG. 28. Rapid identification of HPV types 16 and 18 in human samples byDETECTR. (A) Diagram of HPV16 and HPV18 sequences within thehypervariable loop V of the L1 gene targeted by Cas12a; highlightedbases indicate 5′ PAM sequence. (B) Heatmap represents normalized meanfluorescence values of HPV types 16 and 18 detected in human cell linesby DETECTR; normalized scale represented in (D). (C) Schematic outliningDNA extraction from human anal samples to HPV identification by DETECTR.(D) Identification of HPV types 16 and 18 in 25 patient samples by PCR(left) and DETECTR (right); DETECTR heatmap represents normalized meanfluorescence values.

FIG. 41. Identification of HPV types 16 and 18 in human cell lines andpatient samples by DETECTR. (A) Schematic of HPV detection by DETECTR orCas12a alone. (B) Detection of HPV types 16 or 18 in SiHa (integratedHPV16), HeLa (integrated HPV18) and BJAB (no HPV) human cell lines, withor without RPA amplification. (C) Detection of HPV types 16 or 18 byDETECTR in 25 human anal clinical samples; BJAB cell line (no HPV) usedas a control. Error bars represent mean ±s.d., where n=3 replicates. (D)Plot of 95% confidence intervals of difference between control andsample groups, based on a one-way ANOVA with Dunnett's post test, wheren=3 replicates. Highlighted sample numbers indicate positive detectionof HPV16 (left) or HPV18 (right) in patient samples, where **p≤0.01 and***p≤0.001.

FIG. 42. PCR and hybrid capture validation and genotyping of HPV inhuman clinical samples. (A) Summary of PCR-based detection of HPV types16 (column 2 and yellow circles) and 18 (column 3 and orange circles)and identification of other HPV types by PCR in 25 in patient samples(column 4) (2); subjective intensive values (0-4 scale) were assignedfor each PCR-based validation (columns 2 and 3). (B) Heatmap depictionof PCR results.

Together, these findings support a unifying mechanism of targetinterference that begins with the Cas12-guide RNA complex binding to acomplementary DNA sequence in a PAM-dependent (dsDNA) or PAM-independent(ssDNA) manner (FIG. 29). Within a host bacterium, such enzymeactivation could provide simultaneous protection from both dsDNA andssDNA phages, and could also target ssDNA sequences that arisetemporarily during phage replication or transcription. In agenome-editing context, target-activated ssDNA cutting by Cas12 may be arare event, but it has the potential to cleave transiently exposed ssDNAat replication forks, R-loops and transcription bubbles, or ssDNAtemplates used for homology-directed repair. Finally, unleashing thessDNase activity of Cas12 proteins offers a new strategy to improve thespeed, sensitivity and specificity of nucleic acid detection forpoint-of-care diagnostic applications.

FIG. 29. Model for PAM-dependent and PAM-independent activation of cisand trans-cleavage by Cas12a. The Cas12a-crRNA complex binds to acomplementary dsDNA in a PAM-dependent manner (top) or ssDNA in aPAM-independent manner (bottom), which is sufficient to unleashindiscriminate ssDNase activity by the RuvC nuclease. Cas12 proteins(e.g., Cas12a) can also release their PAM-distal cleavage products,which exposes the RuvC active site for multiple rounds of non-specificssDNA degradation.

TABLE 2 Nucleic acids used in this study. SEQ Name Sequence ID NO. RNALbCas12a crRNA - Target 1 UAAUUUCUACUAAGUGUAGAUCGUC 20 GCCGUCCAGCUCGACCLbCas12a crRNA - pUC19 UAAUUUCUACUAAGUGUAGAUCAAC 21 GUCGUGACUGGGAAAACCCULbCas12a crRNA - M13 UAAUUUCUACUAAGUGUAGAUAACG 22 AACCACCAGCAGAAGALbCas12a crRNA - Target 2 UAAUUUCUACUAAGUGUAGAUGAUC 23 GUUACGCUAACUAUGA LbCas12a crRNA - Target 3 UAAUUUCUACUAAGUGUAGAUCCUG 24 GGUGUUCCACAGCUGALbCas12a crRNA - Plasmid 1 UAAUUUCUACUAAGUGUAGAUCUAC 25AUUACAGGCUAACAAA  LbCas12a crRNA - Plasmid 2 UAAUUUCUACUAAGUGUAGAUGUAC26 AUUGCAAGAUACUAAA LbCas12a crRNA - HPV16-L1 UAAUUUCUACUAAGUGUAGAUUGAA27 GUAGAUAUGGCAGCAC LbCas12a crRNA - HPV18-L1 UAAUUUCUACUAAGUGUAGAUACAA28 UAUGUGCUUCUACACA AsCas12a crRNA - Target 2 UAAUUUCUACUCUUGUAGAUGAUCG29 UUACGCUAACUAUGA FnCas12a crRNA - Target 2 UAAUUUCUACUGUUGUAGAUGAUCG30 UUACGCUAACUAUGA AaCas12b crRNA - Target 2 GUCUAGAGGACAGAAUUUUUCAACGG31 GUGUGCCAAUGGCCACUUUCCAGGUG GCAAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUGGCACGAUCGUUACGCU AACUAUGA SpCas9 sgRNA - Target 1CGUCGCCGUCCAGCUCGACCGUUUU 32 AGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCSpCas9 sgRNA - M13 AACGAACCACCAGCAGAAGAGUUUU 33AGAGCUAUGCUGUUUUGGAAACAAAA CAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGC SpCas9 sgRNA - Target 2GAUCGUUACGCUAACUAUGAGUUUU 34 AGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCNmCas9 sgRNA - Target 2 GAUCGUUACGCUAACUAUGAGUUGU 35AGCUCCCUUUCUCAUUUCGCAGUGCG AAAGCACUGCGAAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAAGAUG UGCCGCAACGCUCUGCCCCUUAAAGC UUCUGCCjCas9 sgRNA - Target 2 GAUCGUUACGCUAACUAUGAGUUUU 36AGUCCCUUUUUAAAUUUCUUUAUGGU AAAAUUAUAAUCUCAUAAGAAAUUUAAAAAGGGACUAAAAUAAAGAGUUU GCGGGACUCUGCGGGGUUACAAUCCC CUAAAACCGCUUTarget 1 ssRNA GCCGGGGUGGUGCCCAUCCUGGUCGA 37 GCUGGACGGCGACGUAAACGGCCACAAGC Target 2 ssRNA UAGCAUUCCACAGACAGCCCUCAUAG 38UUAGCGUAACGAUCUAAAGUUUUGUC GUC DNA non-specific NTSAGCTTGTCTGCCATGGACATGCAGACT 39 ATACTGTTATTGTTGTACAGACCGAAT TCCCnon-specific TS GGGAATTCGGTCTGTACAACAATAACA 40GTATAGTCTGCATGTCCATGGCAGACA AGCT Target l_NTS GCTTGTGGCCGTTTACGTCGCCGTCC 41 AGCTCGACCAGGATGGGCACCACCCC GGC Target l_TSGCCGGGGTGGTGCCCATCCTGGTCGA 42 GCTGGACGGCGACG TAAACGGCCAC AAGCTarget 1_20-19_NTS GCTTGTGGCCGTTTA CGTCGCCGTCC 43AGCTCGAGGAGGATGGGCACCACCCC GGC Target 1_20-19_TSGCCGGGGTGGTGCCCATCCTCCTCGA 44 GCTGGACGGCGACG TAAACGGCCAC AAGCTarget 1_18-17_NTS GCTTGTGGCCGTTTA CGTCGCCGTCC 45AGCTCCTCCAGGATGGGCACCACCCC GGC Target 1_18-17_TSGCCGGGGTGGTGCCCATCCTGGAGGA 46 GCTGGACGGCGACG TAAACGGCCAC AAGCTarget 1_16-15_NTS GCTTGTGGCCGTTTA CGTCGCCGTCC 47AGCAGGACCAGGATGGGCACCACCCC GGC Target 1_16-15_TSGCCGGGGTGGTGCCCATCCTGGTCCT 48 GCTGGACGGCGACG TAAACGGCCAC AAGCTarget 1_14-13_NTS GCTTGTGGCCGTTTA CGTCGCCGTCC 49ACGTCGACCAGGATGGGCACCACCCC GGC Target 1_14-13_TSGCCGGGGTGGTGCCCATCCTGGTCGA 50 CGTGGACGGCGACG TAAACGGCCAC AAGCTarget 1_12-11_NTS GCTTGTGGCCGTTTA CGTCGCCGTCG 51TGCTCGACCAGGATGGGCACCACCCC GGC Target 1_12-11_TSGCCGGGGTGGTGCCCATCCTGGTCGA 52 GCACGACGGCGACG TAAACGGCCAC AAGCTarget 1_10-9_NTS GCTTGTGGCCGTTTA CGTCGCCGAGC 53AGCTCGACCAGGATGGGCACCACCCC GGC Target 1_10-9_TSGCCGGGGTGGTGCCCATCCTGGTCGA 54 GCTGCTCGGCGACG TAAACGGCCAC AAGCTarget 1_8-7_NTS GCTTGTGGCCGTTTA CGTCGCGCTCC 55AGCTCGACCAGGATGGGCACCACCCC GGC Target 1_8-7_TSGCCGGGGTGGTGCCCATCCTGGTCGA 56 GCTGGAGCGCGACG TAAACGGCCAC AAGCTarget 1_6-5_NTS GCTTGTGGCCGTTTA CGTCCGCGTCC 57AGCTCGACCAGGATGGGCACCACCCC GGC Target 1_6-5_TSGCCGGGGTGGTGCCCATCCTGGTCGA 58 GCTGGACGCGGACG TAAACGGCCAC AAGCTarget 1_4-3_NTS GCTTGTGGCCGTTTA CGAGGCCGTCC 59AGCTCGACCAGGATGGGCACCACCCC GGC Target 1_4-3_TSGCCGGGGTGGTGCCCATCCTGGTCGA 60 GCTGGACGGCCTCG TAAACGGCCAC AAGCTarget 1_2-1_NTS GCTTGTGGCCGTTTAGCTCGCCGTCC 61AGCTCGACCAGGATGGGCACCACCCC GGC Target 1_2-1_TSGCCGGGGTGGTGCCCATCCTGGTCGA 62 GCTGGACGGCGAGCTAAACGGCCAC AAGCTarget l_mut-PAM_NTS GCTTGTGGCCGAGCA CGTCGCCGTCC 63AGCTCGACCAGGATGGGCACCACCCC GGC Target l_mut-PAM_TSGCCGGGGTGGTGCCCATCCTGGTCGA 64 GCTGGACGGCGACG TGCTCGGCCACA AGCTarget 1_5nt_TS CGACG TAAACGGCCACAAGC 65 Target 1_10nt_TS GACGGCGACGTAAACGGCCACAAGC 66 Target 1_15nt_TS AGCTGGACGGCGACG TAAACGGCCA 67 CAAGCTarget 1_20nt_TS GGTCGAGCTGGACGGCGACG TAAAC 68 GGCCACAAGCTarget 1_25nt_TS ATCCTGGTCGAGCTGGACGGCGACG 69 TAAACGGCCACAAGCTarget 1_5nt_NTS GCTTGTGGCCGTTTA CGTCG 70 Target 1_10nt_NTSGCTTGTGGCCGTTTA CGTCGCCGTC 71 Target 1_15nt_NTS GCTTGTGGCCGTTTACGTCGCCGTCC 72 AGCT Target 1_20nt_NTS GCTTGTGGCCGTTTA CGTCGCCGTCC 73AGCTCGACC Target 1_25nt_NTS GCTTGTGGCCGTTTA CGTCGCCGTCC 74AGCTCGACCAGGAT Target 2_NTS GACGACAAAACTTTA GATCGTTACGC 75TAACTATGAGGGCTGTCTGTGGAATG CTA Target 2_TS TAGCATTCCACAGACAGCCCTCATAGT76 TAGCGTAACGATC TAAAGTTTTGTCG TC Target 2_20-19_NTS GACGACAAAACTTTAGATCGTTACGC 77 TAACTATCTGGGCTGTCTGTGGAATGC TA Target 2_20-19_TSTAGCATTCCACAGACAGCCCAGATAG 78 TTAGCGTAACGATC TAAAGTTTTGTC GTCTarget 2_18-17_NTS GACGACAAAACTTTA GATCGTTACGC 79TAACTTAGAGGGCTGTCTGTGGAATG CTA Target 2_18-17_TSTAGCATTCCACAGACAGCCCTCTAAGT 80 TAGCGTAACGATC TAAAGTTTTGTCG TCTarget 2_16-15_NTS GACGACAAAACTTTA GATCGTTACGC 81TAAGAATGAGGGCTGTCTGTGGAATG CTA Target 2_16-15_TSTAGCATTCCACAGACAGCCCTCATTCT 82 TAGCGTAACGATC TAAAGTTTTGTCG TCTarget 2_14-13_NTS GACGACAAAACTTTA GATCGTTACGC 83TTTCTATGAGGGCTGTCTGTGGAATGC TA Target 2_14-13_TSTAGCATTCCACAGACAGCCCTCATAG 84 AAAGCGTAACGATC TAAAGTTTTGTC GTCTarget 2_12-11_NTS GACGACAAAACTTTA GATCGTTACGG 85AAACTATGAGGGCTGTCTGTGGAATG CTA Target 2_12-11_TSTAGCATTCCACAGACAGCCCTCATAGT 86 TTCCGTAACGATC TAAAGTTTTGTCGT CTarget 2_10-9_NTS GACGACAAAACTTTA GATCGTTAGCC 87TAACTATGAGGGCTGTCTGTGGAATG CTA Target 2_10-9_TSTAGCATTCCACAGACAGCCCTCATAGT 88 TAGGCTAACGATC TAAAGTTTTGTCG TCTarget 2_8-7_NTS GACGACAAAACTTTA GATCGTATCGC 89TAACTATGAGGGCTGTCTGTGGAATG CTA Target 2_8-7_TSTAGCATTCCACAGACAGCCCTCATAGT 90 TAGCGATACGATC TAAAGTTTTGTCG TCTarget 2_6-5_NTS GACGACAAAACTTTA GATCCATACGC 91TAACTATGAGGGCTGTCTGTGGAATG CTA Target 2_6-5_TSTAGCATTCCACAGACAGCCCTCATAGT 92 TAGCGTATGGATC TAAAGTTTTGTCG TCTarget 2_4-3_NTS GACGACAAAACTTTA GAAGGTTACGC 93TAACTATGAGGGCTGTCTGTGGAATG CTA Target 2_4-3_TSTAGCATTCCACAGACAGCCCTCATAGT 94 TAGCGTAACCTTC TAAAGTTTTGTCGT CTarget 2_2-1_NTS GACGACAAAACTTTACTTCGTTACGC 95TAACTATGAGGGCTGTCTGTGGAATG CTA Target 2_2-1_TSTAGCATTCCACAGACAGCCCTCATAGT 96 TAGCGTAACGAAGTAAAGTTTTGTCG TCTarget 2_mut-PAM_NTS GACGACAAAACAGCA GATCGTTACGC 97TAACTATGAGGGCTGTCTGTGGAATG CTA Target 2_mut-PAM_TSTAGCATTCCACAGACAGCCCTCATAGT 98 TAGCGTAACGATC TGCTGTTTTGTCGT CTarget 2_NmCas9_NTS GACGACAAAACTTTAGATCGTTACGC 99 TAACTATGAGGGCGAGTTGTGGAATG CTA Target 2_NmCas9_TS TAGCATTCCACAACTCGCCC TCATAGT100 TAGCGTAACGATCTAAAGTTTTGTCG TC Target 2_CjCas9_TSGACGACAAAACTTTAGATCGTTACGC 101 TAACTATGA GGGCCAAATGTGGAATG CTATarget 2_CjCas9_TS TAGCATTCCACATTTGGCCC TCATAGT 102TAGCGTAACGATCTAAAGTTTTGTCG TC Target 3_NTS AGTTGTGTTAGTTTA CCTGGGTGTTCC103 ACAGCTGATAGTGATTGCCTTGAATA AA Target 3_TSTTTATTCAAGGCAATCACTATCAGCTG 104 TGGAACACCCAGG TAAACTAACACAA CTTarget 3_20-19_NTS AGTTGTGTTAGTTTA CCTGGGTGTTCC 105ACAGCTCTTAGTGATTGCCTTGAATAA A Target 3_20-19_TSTTTATTCAAGGCAATCACTAAGAGCTG 106 TGGAACACCCAGG TAAACTAACACAA CTTarget 3_18-17_NTS AGTTGTGTTAGTTTA CCTGGGTGTTCC 107ACAGGAGATAGTGATTGCCTTGAATA AA Target 3_18-17_TSTTTATTCAAGGCAATCACTATCTCCTG 108 TGGAACACCCAGG TAAACTAACACAA CTTarget 3_16-15_NTS AGTTGTGTTAGTTTA CCTGGGTGTTCC 109ACTCCTGATAGTGATTGCCTTGAATAA A Target 3_16-15_TSTTTATTCAAGGCAATCACTATCAGGAG 110 TGGAACACCCAGG TAAACTAACACAA CTTarget 3_14-13_NTS AGTTGTGTTAGTTTA CCTGGGTGTTCC 111TGAGCTGATAGTGATTGCCTTGAATA AA Target 3_14-13_TSTTTATTCAAGGCAATCACTATCAGCTC 112 AGGAACACCCAGG TAAACTAACACAA CTTarget 3_12-11_NTS AGTTGTGTTAGTTTA CCTGGGTGTTG 113GACAGCTGATAGTGATTGCCTTGAAT AAA Target 3_12-11_TSTTTATTCAAGGCAATCACTATCAGCTG 114 TCCAACACCCAGG TAAACTAACACAA CTTarget 3_10-9_NTS AGTTGTGTTAGTTTA CCTGGGTGAAC 115CACAGCTGATAGTGATTGCCTTGAAT AAA Target 3_10-9_TSTTTATTCAAGGCAATCACTATCAGCTG 116 TGGTTCACCCAGG TAAACTAACACAA CTTarget 3_8-7_NTS AGTTGTGTTAGTTTA CCTGGGACTTCC 117ACAGCTGATAGTGATTGCCTTGAATA AA Target 3_8-7_TSTTTATTCAAGGCAATCACTATCAGCTG 118 TGGAAGTCCCAGG TAAACTAACACAA CTTarget 3_6-5_NTS AGTTGTGTTAGTTTA CCTGCCTGTTCC 119ACAGCTGATAGTGATTGCCTTGAATA AA Target 3_6-5_TSTTTATTCAAGGCAATCACTATCAGCTG 120 TGGAACAGGCAGG TAAACTAACACA ACTTarget 3_4-3_NTS AGTTGTGTTAGTTTA CCACGGTGTTCC 121ACAGCTGATAGTGATTGCCTTGAATA AA Target 3_4-3_TSTTTATTCAAGGCAATCACTATCAGCTG 122 TGGAACACCGTGG TAAACTAACACAA CTTarget 3_2-1_NTS AGTTGTGTTAGTTTAGGTGGGTGTTC 123CACAGCTGATAGTGATTGCCTTGAAT AAA Target 3_2-1_TSTTTATTCAAGGCAATCACTATCAGCTG 124 TGGAACACCCACCTAAACTAACACAA CTTarget 3_mut-PAM_NTS AGTTGTGTTAGAGCA CCTGGGTGTTC 125CACAGCTGATAGTGATTGCCTTGAAT AAA Target 3_mut-PAM_TSTTTATTCAAGGCAATCACTATCAGCTG 126 TGGAACACCCAGG TGCTCTAACACAA CTFQ substrates ssDNA-FQ reporter /56-FAM/TTATT/3IABkFQ/ ssRNA-FQ reporter/56-FAM/rUrUrArUrU/3IABkFQ/ Dnase-Alert substrate (IDT) proprietaryRPA primers Plasmid 1_F GCAAACCACCTATAGGGGAACAC 127 Plasmid 1_RCAGCCAACTCAGCTTCCTTTC 128 Plasmid 2_F CATGCCGCCACGTCTAATGTTTC 129Plasmid 2_R GGTGAAGCACGCATACCTGTG 130 HPV16-L1_FTTGTTGGGGTAACCAACTATTTGTTAC 131 TGTT HPV16-L1_RCCTCCCCATGTCTGAGGTACTCCTTAA 132 AG HPV18-L1_FGCATAATCAATTATTTGTTACTGTGGT 133 AGATACCACT HPV18-L1-RGCTATACTGCTTAAATTTGGTAGCATC 134 ATATTGC

Materials and Methods

Protein expression and purification. DNA sequences encoding SpCas9 andCas12 proteins and mutants were cloned into a custom pET-basedexpression vector containing an N-terminal 10×His-tag, maltose-bindingprotein (MBP) and TEV protease cleavage site. Point mutations wereintroduced by around-the-horn PCR and verified by DNA sequencing.Proteins were purified as described, with the following modifications:E. coli BL21(DE3) containing SpCas9 or Cas12 expression plasmids weregrown in Terrific Broth at 16° C. for 14 hr. Cells were harvested andresuspended in Lysis Buffer (50 mM Tris-HC1, pH 7.5, 500 mM NaCl, 5%(v/v) glycerol, 1 mM TCEP, 0.5 mM PMSF and 0.25 mg/ml lysozyme),disrupted by sonication, and purified using Ni-NTA resin. Afterovernight TEV cleavage at 4° C., proteins were purified over an MBPTrapHP column connected to a HiTrap Heparin HP column for cation exchangechromatography. The final gel filtration step (Superdex 200) was carriedout in elution buffer containing 20 mM Tris-HC1, pH 7.5, 200 mM NaCl (or250 mM NaCl for AaCas12b), 5% (v/v) glycerol and 1 mM TCEP. All proteinstested in this study are shown in FIG. 30.

Nucleic acid preparation. DNA substrates were synthesized commercially(IDT). For FQ-reporter assays, activator DNA duplexes were prepared byannealing 5-fold molar excess of the NTS to TS in lx hybridizationbuffer (20 nM Tris-C1, pH 7.5, 100 mM KCl, 5 mM MgCl₂), heating at 95°C. and slow-cooling on the benchtop. HPV16 and HPV18 fragments weresynthesized as gBlocks (IDT) and cloned into a custom pET-based vectorvia Gibson assembly. Plasmid DNA for titration experiments wasquantified using a Qubit fluorometer (Invitrogen). For radiolabeledcleavage assays, PAGE-purified DNA oligos were prepared as described.

sgRNA templates were PCR amplified from a pUC19 vector or overlappingprimers containing a T7 promoter, 20 nucleotide target sequence and ansgRNA scaffold. The amplified PCR product served as the DNA template forin vitro transcription reactions, which were performed as described.crRNAs were transcribed in vitro using a single-stranded DNA templatecontaining a T7 promoter, repeat and spacer in the reverse complementorientation, which was annealed to T7 forward primer in lx hybridizationbuffer. All DNA and RNA substrates are listed in Table S1.

DNA cleavage assays. Generally, Cas12a-mediated cleavage assays werecarried out in cleavage buffer consisting of 20 mM HEPES (pH 7.5), 150mM KCl, 10 mM MgCl₂, 1% glycerol and 0.5 mM DTT. For M13-targetingassays, 30 nM Cas12a was pre-assembled with either 36 nM ofM13-targeting crRNA (cis) or with 36 nM of crRNA and 40 nM complementaryssDNA (activator) with no sequence homology to M13 (trans) at 37° C. for10 min. The reaction was initiated by adding 10 nM Ml3mpl8 ssDNA (NewEngland Biolabs) and incubated at 37° C. for indicated timepoints.Reactions were quenched with DNA loading buffer (30% (v/v) glycerol,0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol) containing 15mM EDTA and separated by 1.5% agarose gel pre-stained with SYBER Gold(Invitrogen).

For radiolabeled cleavage assays, the substrates used were5′-end-labeled with T4 PNK (NEB) in the presence of gamma ³²P-ATP. FordsDNA substrates, the non-target strand was first 5′-end-labeled andthen annealed with excess corresponding target strand. Theconcentrations of Cas12a (or SpCas9), guide RNA and ³²P-labeledsubstrates used in the reaction were 30 nM, 36 nM and 1-3 nM (unlessotherwise stated), respectively. Reactions were incubated for 30 min(unless otherwise stated) at 37° C. (or 47.5° C. for the thermophilicAacCasl2b) and quenched with formamide loading buffer (finalconcentration 45% formamide and 15 mM EDTA, with trace amount of xylenecyanol and bromophenol blue) for 2-3 min at 90° C. The substrates andproducts were resolved by 12% urea-denaturing PAGE gel and quantifiedwith Amersham Typhoon (GE Healthcare).

For substrate turnover studies, the pre-assembled Cas12a-crRNA orCas12a-crRNA-activator (target ssDNA or dsDNA) were incubated at 37° C.for 10 min, and 30 nM of the pre-assembled RNP were used for eachreaction with various substrate concentrations at 15, 30, 45, and 60 nM,respectively.

Fluorophore quencher (FQ)-labeled reporter assays. LbCas12a-crRNAcomplexes were pre-assembled by incubating 200 nM LbCpf1 with 250 nMcrRNA and 4 nM activator (ssDNA, dsDNA or ssRNA) at 37° C. for 30 min.The reaction was initiated by diluting LbCas12a complexes to a finalconcentration of 50 nM LbCas12a: 62.5 nM crRNA: 1 nM activator in asolution containing 1×Binding Buffer (20 mM Tris-HC1, pH 7.5, 100 mMKC1, 5 mM MgCl₂, 1 mM DTT, 5% glycerol, 50 μg ml⁻¹ heparin) and 50 nMDNaseAlert substrate™ (IDT) or custom ssDNA/ssRNA FQ reporter substratesin a 20 μl reaction (Table S1). HPV detection assays were performed asabove, with the following modifications: LbCas12a was pre-assembled withan HPV16 or HPV18-targeting crRNA and diluted in a solution containing1×Binding Buffer, custom ssDNA-FQ reporter and 1, 10, 100, or 1000 nM ofHPV16- or HPV18-containing plasmids. Reactions (20 μl, 384-wellmicroplate format) were incubated in a fluorescence plate reader (TecanInfinite Pro F200) for up to 120 minutes at 37° C. with fluorescencemeasurements taken every 30 seconds (DNaseAlert substrate=λ_(ex): 535nm; λ_(em): 595 nm, custom ssDNA/ssRNA FQ substrates=λ_(ex): 485 nm;λ_(em): 535 nm).

For trans-cleavage rate determination, background-corrected fluorescencevalues were calculated by subtracting fluorescence values obtained fromreactions carried out in the absence of target plasmid. The resultingdata were fit to a single exponential decay curve (GraphPad Software),according to the following equation: Fraction cleaved =A×(1−exp(−k×t)),where A is the amplitude of the curve, k is the first-order rateconstant, and t is time.

For Michaelis-Menten analysis, LbCas12a-crRNA-activator (target ssDNA ordsDNA) complexes were prepared as described above, and reaction wasinitiated by diluting LbCas12a complexes to a final concentration of 5nM LbCas12a: 6.25 nM crRNA: 0.1 nM activator (effective complex =0.1 nM)in a solution containing 1× Binding Buffer and 0.001, 0.01, 0.1, 0.2,0.5, 1 or 2 uM of DNaseAlert™ substrate (IDT). Reactions were incubatedin a fluorescence plate reader for up to 30 minutes at 37° C. withfluorescence measurements taken every 30 seconds (λ_(ex): 535 nm;λ_(em): 595 nm). The initial velocity (V₀) was calculated by fitting toa linear regression and plotted against the substrate concentration todetermine the Michaelis-Menten constants (GraphPad Software), accordingto the following equation: Y=(V_(max)×X)/(K_(m)+X), where X is thesubstrate concentration and Y is the enzyme velocity. The turnovernumber (k_(cat)) was determined by the following equation:k_(cat)=V_(max)/E_(t), where E_(t)=0.1 nM.

Human clinical sample collection and DNA preparation. Anal sample donorswere recruited from the UCSF Anal Neoplasia Clinic, Research andEducation Center (ANCRE). The study was approved by the UCSF Committeeon Human Research. After informed consent was obtained, participants hadan anal swab inserted into a ThinprepTM vial for anal cytology and HPVtesting. Cell suspension left over from the first swab after monolayercytology slides were made was used for HPV DNA PCR.

A crude DNA preparation was made by pelleting 1.5 ml of the cellsuspension. After the pellet was allowed to dry, it was suspended in 100μl Tris-EDTA with proteinase K (Life Technologies) at a concentration of200 μg/ml and incubated at 56° C. for 1 hour, then the proteinase K washeat inactivated. Five μl of this was used in the HPV consensus PCR. DNApreparation from human cell lines (SiHa, HeLa, BJAB) was performed asabove, with the following modifications: 10⁶-10⁷ cells were harvested,resuspended in 100 μl Tris-EDTA with proteinase K, incubated at 56° C.for 1 hour, then the proteinase K was heat inactivated. One μl of thissample was used for DETECTR experiments.

DETECTR assays. DETECTR combined Recombinase Polymerase Amplification(RPA) using TwistAmp Basic (Twist Biosciences) followed by Cas12adetection in the same reaction. Briefly, 50 μl reactions containing 1 μlsample, 0.48 μM forward and reverse primer, 1× rehydration buffer, 14 mMmagnesium acetate and RPA mix were incubated at 37° C. for 10 minutes.The RPA reaction (18 μl) was transferred to a 384-well microplate and 50nM LbCas12a: 62.5 nM crRNA: 50 nM custom ssDNA-FQ reporter was addeddirectly to the reaction (20 μl final volume). Reactions were incubatedin a fluorescence plate reader (Tecan Infinite Pro F200) for 1-2 h at37° C. with fluorescence measurements taken every minute (λ_(ex): 485nm; λ_(cm): 535 nm).

For HPV identification by DETECTR, detection values of HPV types 16 or18 in human samples were normalized to the maximum mean fluorescencesignal obtained using the HPV16-or HPV18-targeting crRNA, respectively.A one-way ANOVA with Dunnett's post test was used to determine thepositive cutoff (set at p≤0.05) for identification of HPV16 or HPV18 inpatient samples. Based on this cutoff, 100% of samples were accuratelyidentified for HPV16 infection (25/25 agreement with PCR-based results),while 92% of samples were accurately identified for HPV18 infection(23/25 agreement with PCR-based results).

HPV genotyping and validation. PCR was performed as described previouslyusing a modified pool of MY09/MY11 consensus HPV L1 primers as well asprimers for amplification of the human beta-globin as an indicator ofspecimen adequacy as described previously. After 40 amplificationcycles, specimens were probed with a biotin-labeled HPV L1 consensusprobe mixture. A separate membrane was probed with a biotin-labeledprobe to the human beta-globin gene. Specimens were typed by hybridizingto 38 different HPV types, 6/11, 16, 18, 26/69, 30, 31, 32/42, 33, 34,35, 39, 45, 51, 52, 53, 54, 56, 57/2/27, 58, 59, 61, 62, 66, 67, 68, 70,71, 72, 73, 81, 82, 83, 84, 85, 86/87, 90/106, 97, 102/89, as well astwo separate mixtures. Mix1 contains 7, 13, 40, 43, 44, 55, 74, and 91,and Mix 2 contains 3, 10, 28, 29, 77, 78, and 94. Specimens negative forbeta-globin gene amplification were excluded from analysis. The resultsof PCR were recorded on a scale from 0 to 5 based on the intensity ofthe signal on the dot-blots, as described previously. Samples withresults recorded as 1 or more were considered to be positive.

Example 4 Trans-Cleavage Activity of Cas12d and Cas12e

trans-cleavage activity was demonstrated for two additional type VCRISPR/Cas effector proteins, CasX (Cas12e) and CasY (Cas12d) using aDETECTR assay (FIG. 43).

Example 5 Identification of a Single Nucleotide Polymorphism (SNP)within the HERC2 gene Responsible for Brown or Blue Eyes

DETECTR was used to detect eye color SNPs from saliva samples usingCas12a (FIG. 44). Sample preparation: 500 μL of phosphate bufferedsaline was added to ˜500 μL of volunteer saliva and centrifuged for 5min at 1800 g. The supernatant was decanted and the pellet wasresuspended in 100 μL phosphate buffered saline with 0.2% Triton X-100before incubation at 95° C. for 5 min. 1 μL of sample was used as directinput into RPA reactions. The following nucleic acids were used forthese experiments:

RPA primers: F primer: (SEQ ID NO: 153) CAAAGAGAAGCCTCGGCC R primer:(SEQ ID NO: 154) GTGTTAATACAAAGGTACAGGAACAAAGAATTTG HERC2 G-SNP crRNA:(SEQ ID NO: 155) GTAATTTCTACTAAGTGTAGATAGCATTAAGTGTCAAGTTCTHERC2 A-SNP crRNA: (SEQ ID NO: 156)GTAATTTCTACTAAGTGTAGATAGCATTAAATGTCAAGTTCT

Example 6 Identification of the X or Y Chromosomes through Detection ofthe XIST (within X chromosome) or SRY (within Y chromosome) Genes fromHuman Saliva

FIG. 45 provides data demonstrating the identification of the X or Ychromosomes through detection of the XIST (within X chromosome) or SRY(within Y chromosome) genes from human saliva. The following nucleicacids were used for these experiments:

XIST crRNA: (SEQ ID NO: 157) GTAATTTCTACTAAGTGTAGATACTAGTCCCTTGTACTGATASRY crRNA: (SEQ ID NO: 158) GTAATTTCTACTAAGTGTAGATGCATTCTGGGATTCTCTAGAXIST RPA primers: F primer: (SEQ ID NO: 159)CTATCTGAATGAATTGATTTGGGGCTTG R primer: (SEQ ID NO: 160)GCAATGTCAAAATCGCCATTTTAAGC SRY RPA primers: F primer: (SEQ ID NO: 161)AGGCAACGTCCAGGATAGAGTG R primer: (SEQ ID NO: 162)CAGTAAGCATTTTCCACTGGTATCCCAG

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of detecting a target DNA in a sample,the method comprising: (a) contacting the sample with: (i) a type VCRISPR/Cas effector protein; (ii) a guide RNA comprising: a region thatbinds to the type V CRISPR/Cas effector protein, and a guide sequencethat hybridizes with the target DNA; and (iii) a detector DNA that issingle stranded and does not hybridize with the guide sequence of theguide RNA; and (b) measuring a detectable signal produced by cleavage ofthe single stranded detector DNA by the type V CRISPR/Cas effectorprotein, thereby detecting the target DNA.
 2. The method of claim 1,comprising contacting the sample with a precursor guide RNA array,wherein the type V CRISPR/Cas effector protein cleaves the precursorguide RNA array to produce said guide RNA and at least one additionalguide RNA.
 3. The method of claim 1 or claim 2, wherein the target DNAis single stranded.
 4. The method of claim 1 or claim 2, wherein thetarget DNA is double stranded.
 5. The method of any one of claims 1-4,wherein the target DNA is viral DNA.
 6. The method of any one of claims1-4, wherein the target DNA is papovavirus, hepdnavirus, herpesvirus,adenovirus, poxvirus, or parvovirus DNA.
 7. The method of any one ofclaims 1-4, wherein the type V CRISPR/Cas effector protein is a Cas12protein.
 8. The method of any one of claims 1-6, wherein the type VCRISPR/Cas effector protein is a Cas12a (Cpf1) or Cas12b (C2c1) protein.9. The method according to any one of claims 1-8, wherein the samplecomprises DNA molecules from a cell lysate.
 10. The method according toany one of claims 1-9, wherein the sample comprises cells.
 11. Themethod according to any one of claims 1-10, wherein said contacting iscarried out inside of a cell in vitro, ex vivo, or in vivo.
 12. Themethod according to claim 11, wherein the cell is a eukaryotic cell. 13.The method according to any one of claims 1-12, wherein the target DNAcan be detected at a concentration as low as 200 fM.
 14. The methodaccording to any one of claims 1-13, comprising determining an amount ofthe target DNA present in the sample.
 15. The method according to claim14, wherein said determining comprises: measuring the detectable signalto generate a test measurement; measuring a detectable signal producedby a reference sample or cell to generate a reference measurement; andcomparing the test measurement to the reference measurement to determinean amount of target DNA present in the sample.
 16. The method accordingto any one of claims 1-15, wherein measuring a detectable signalcomprises one or more of: gold nanoparticle based detection,fluorescence polarization, colloid phase transition/dispersion,electrochemical detection, and semiconductor-based sensing.
 17. Themethod according to any one of claims 1-16, wherein the single strandeddetector DNA comprises a fluorescence-emitting dye pair.
 18. The methodaccording to claim 17, wherein the fluorescence-emitting dye pairproduces an amount of detectable signal prior to cleavage of the singlestranded detector DNA, and the amount of detectable signal is reducedafter cleavage of the single stranded detector DNA.
 19. The methodaccording to claim 17, wherein the single stranded detector DNA producesa first detectable signal prior to being cleaved and a second detectablesignal after cleavage of the single stranded detector DNA.
 20. Themethod according to any one of claims 17-19, wherein thefluorescence-emitting dye pair is a fluorescence resonance energytransfer (FRET) pair.
 21. The method according to claim 17, wherein anamount of detectable signal increases after cleavage of the singlestranded detector DNA.
 22. The method according to claim 17 or claim 21,wherein the fluorescence-emitting dye pair is a quencher/fluor pair. 23.The method according to any one of claims 17-22, wherein the singlestranded detector DNA comprises two or more fluorescence-emitting dyepairs.
 24. The method according to claim 23, wherein said two or morefluorescence-emitting dye pairs include a fluorescence resonance energytransfer (FRET) pair and a quencher/fluor pair.
 25. The method accordingto any one of claims 1-24, wherein the single stranded detector DNAcomprises a modified nucleobase, a modified sugar moiety, and/or amodified nucleic acid linkage.
 26. A kit for detecting a target DNA in asample, the kit comprising: (a) a guide RNA, or a nucleic acid encodingthe guide RNA, or a precursor guide RNA array comprising the guide RNA,or a nucleic acid encoding the precursor guide RNA array; wherein theguide RNA comprises: a region that binds to a type V CRISPR/Cas effectorprotein, and a guide sequence that is complementary to a target DNA; and(b) a labeled detector DNA that is single stranded and does nothybridize with the guide sequence of the guide RNA.
 27. The kit of claim26, further comprising a type V CRISPR/Cas effector protein.
 28. The kitof claim 27, wherein the type V CRISPR/Cas effector protein is a Cas12protein.
 29. The kit of claim 27, wherein the type V CRISPR/Cas effectorprotein is a Cas12a (Cpf1) or Cas12b (C2c1) protein.
 30. The kit of anyone of claims 26-29, wherein the single stranded detector DNA comprisesa fluorescence-emitting dye pair.
 31. The kit of claim 30, wherein thefluorescence-emitting dye pair is a FRET pair.
 32. The kit of claim 30,wherein the fluorescence-emitting dye pair is a quencher/fluor pair. 33.The kit of any one of claims 30-32, wherein the single stranded detectorDNA comprises two or more fluorescence-emitting dye pairs.
 34. The kitof claim 33, wherein said two or more fluorescence-emitting dye pairsinclude a first fluorescence-emitting dye pair that produces a firstdetectable signal and a second fluorescence-emitting dye pair thatproduces a second detectable signal.
 35. A method of cleaving singlestranded DNAs (ssDNAs), the method comprising: contacting a populationof nucleic acids, wherein said population comprises a target DNA and aplurality of non-target ssDNAs, with: (i) a type V CRISPR/Cas effectorprotein; and (ii) a guide RNA comprising: a region that binds to thetype V CRISPR/Cas effector protein, and a guide sequence that hybridizeswith the target DNA, wherein the type V CRISPR/Cas effector proteincleaves non-target ssDNAs of said plurality.
 36. The method of claim 35,comprising contacting the sample with a precursor guide RNA array,wherein the type V CRISPR/Cas effector protein cleaves the precursorguide RNA array to produce said guide RNA and at least one additionalguide RNA.
 37. The method of claim 35 or claim 36, wherein saidcontacting is inside of a cell in vitro, ex vivo, or in vivo.
 38. Themethod of claim 37, wherein the cell is a eukaryotic cell.
 39. Themethod of claim 38, wherein the eukaryotic cell is a plant cell.
 40. Themethod of any one of claims 37-39, wherein the non-target ssDNAs areforeign to the cell.
 41. The method of claim 40, wherein the non-targetssDNAs are viral DNAs.
 42. The method of any one of claims 35-41,wherein the target DNA is single stranded.
 43. The method of any one ofclaims 35-41, wherein the target DNA is double stranded.
 44. The methodof any one of claims 35-43, wherein the target DNA is viral DNA.
 45. Themethod of any one of claims 35-43, wherein the target DNA ispapovavirus, hepdnavirus, herpesvirus, adenovirus, poxvirus, orparvovirus DNA.